Muscle targeting complexes and uses thereof for treating myotonic dystrophy

ABSTRACT

Aspects of the disclosure relate to complexes comprising a muscle-targeting agent covalently linked to a molecular payload. In some embodiments, the muscle-targeting agent specifically binds to an internalizing cell surface receptor on muscle cells. In some embodiments, the molecular payload inhibits expression or activity of a DMPK allele comprising a disease-associated-repeat. In some embodiments, the molecular payload is an oligonucleotide, such as an antisense oligonucleotide or RNAi oligonucleotide.

RELATED APPLICATION

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application No. 63/220,144, filed Jul. 9, 2021,entitled “MUSCLE TARGETING COMPLEXES AND USES THEREOF FOR TREATINGMYOTONIC DYSTROPHY,” which is hereby incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present application relates to targeting complexes for deliveringmolecular payloads (e.g., oligonucleotides) to cells and uses thereof,particularly uses relating to treatment of disease.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing(D082470068US01-SEQ-ZJG.xml; Size: 811,850 bytes; and Date of Creation:Jul. 1, 2022) is herein incorporated by reference in its entirety.

BACKGROUND OF INVENTION

Myotonic dystrophy (DM) is a dominantly inherited genetic disease thatis characterized by myotonia, muscle loss or degeneration, diminishedmuscle function, insulin resistance, cardiac arrhythmia, smooth muscledysfunction, and neurological abnormalities. DM is the most common formof adult-onset muscular dystrophy, with a worldwide incidence of about 1in 8000 people worldwide. Two types of the disease, myotonic dystrophytype 1 (DM1) and myotonic dystrophy type 2 (DM2), have been described.DM1, the more common form of the disease, results from a repeatexpansion of a CTG trinucleotide repeat in the 3′ non-coding region ofDMPK on chromosome 19; DM2 results from a repeat expansion of a CCTGtetranucleotide repeat in the first intron of ZNF9 on chromosome 3. InDM1 patients, the repeat expansion of a CTG trinucleotide repeat, whichmay comprise greater than ˜50 to ˜3,000+ total repeats, leads togeneration of toxic RNA repeats capable of forming hairpin structuresthat bind essential intracellular proteins, e.g. muscleblind-likeproteins, with high affinity resulting in protein sequestration and theloss-of-function phenotypes that are characteristic of the disease.Apart from supportive care and treatments to address the symptoms of thedisease, no effective therapeutic for DM1 is currently available.

SUMMARY OF INVENTION

In some aspects, the disclosure provides complexes that target musclecells for purposes of delivering molecular payloads to those cells. Insome embodiments, complexes provided herein are particularly useful fordelivering molecular payloads that inhibit the expression or activity ofa DMPK allele comprising an expanded disease-associated-repeat, e.g., ina subject having or suspected of having myotonic dystrophy. Accordingly,in some embodiments, complexes provided herein comprise muscle-targetingagents (e.g., muscle targeting antibodies) that specifically bind toreceptors on the surface of muscle cells for purposes of deliveringmolecular payloads to the muscle cells. In some embodiments, thecomplexes are taken up into the cells via a receptor mediatedinternalization, following which the molecular payload may be releasedto perform a function inside the cells. For example, complexesengineered to deliver oligonucleotides may release the oligonucleotidessuch that the oligonucleotides can inhibit mutant DMPK expression in themuscle cells. In some embodiments, the oligonucleotides are released byendosomal cleavage of covalent linkers connecting oligonucleotides andmuscle-targeting agents of the complexes.

One aspect of the present disclosure relates to a complex comprising ananti-transferrin receptor (TfR) antibody covalently linked to amolecular payload configured for reducing expression or activity ofDMPK, wherein the anti-TfR antibody comprises:

(i) a heavy chain variable region (VH) comprising an amino acid sequenceat least 95% identical to SEQ ID NO: 76; and/or a light chain variableregion (VL) comprising an amino acid sequence at least 95% identical toSEQ ID NO: 75;

(ii) a heavy chain variable region (VH) comprising an amino acidsequence at least 95% identical to SEQ ID NO: 69; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 95%identical to SEQ ID NO: 70;

(iii) a heavy chain variable region (VH) comprising an amino acidsequence at least 95% identical to SEQ ID NO: 71; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 95%identical to SEQ ID NO: 70;

(iv) a heavy chain variable region (VH) comprising an amino acidsequence at least 95% identical to SEQ ID NO: 72; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 95%identical to SEQ ID NO: 70;

(v) a heavy chain variable region (VH) comprising an amino acid sequenceat least 95% identical to SEQ ID NO: 73; and/or a light chain variableregion (VL) comprising an amino acid sequence at least 95% identical toSEQ ID NO: 74;

(vi) a heavy chain variable region (VH) comprising an amino acidsequence at least 95% identical to SEQ ID NO: 73; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 95%identical to SEQ ID NO: 75;

(vii) a heavy chain variable region (VH) comprising an amino acidsequence at least 95% identical to SEQ ID NO: 76; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 95%identical to SEQ ID NO: 74;

(viii) a heavy chain variable region (VH) comprising an amino acidsequence at least 95% identical to SEQ ID NO: 77; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 95%identical to SEQ ID NO: 78;

(ix) a heavy chain variable region (VH) comprising an amino acidsequence at least 95% identical to SEQ ID NO: 79; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 95%identical to SEQ ID NO: 80; or

(x) a heavy chain variable region (VH) comprising an amino acid sequenceat least 95% identical to SEQ ID NO: 77; and/or a light chain variableregion (VL) comprising an amino acid sequence at least 95% identical toSEQ ID NO: 80.

In some embodiments, the antibody comprises:

(i) a VH comprising the amino acid sequence of SEQ ID NO: 76 and a VLcomprising the amino acid sequence of SEQ ID NO: 75;

(ii) a VH comprising an amino acid sequence of SEQ ID NO: 69 and a VLcomprising an amino acid sequence of SEQ ID NO: 70;

(iii) a VH comprising an amino acid sequence of SEQ ID NO: 71 and a VLcomprising an amino acid sequence of SEQ ID NO: 70;

(iv) a VH comprising an amino acid sequence of SEQ ID NO: 72 and a VLcomprising an amino acid sequence of SEQ ID NO: 70;

(v) a VH comprising an amino acid sequence of SEQ ID NO: 73 and a VLcomprising an amino acid sequence of SEQ ID NO: 74;

(vi) a VH comprising an amino acid sequence of SEQ ID NO: 73 and a VLcomprising an amino acid sequence of SEQ ID NO: 75;

(vii) a VH comprising an amino acid sequence of SEQ ID NO: 76 and a VLcomprising an amino acid sequence of SEQ ID NO: 74;

(viii) a VH comprising an amino acid sequence of SEQ ID NO: 77 and a VLcomprising an amino acid sequence of SEQ ID NO: 78;

(ix) a VH comprising an amino acid sequence of SEQ ID NO: 79 and a VLcomprising an amino acid sequence of SEQ ID NO: 80; or

(x) a VH comprising an amino acid sequence of SEQ ID NO: 77 and a VLcomprising an amino acid sequence of SEQ ID NO: 80.

In some embodiments, the antibody is selected from the group consistingof a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment, an scFv, an Fv,and a full-length IgG. In some embodiments, the antibody is a Fabfragment.

In some embodiments, the antibody comprises:

(i) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 101; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 90;

(ii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 97; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 85;

(iii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 98; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 85;

(iv) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 99; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 85;

(v) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 100; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 89;

(vi) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 100; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 90;

(vii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 101; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 89;

(viii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 102; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 93;

(ix) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 103; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 95; or

(x) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 102; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 95.

In some embodiments, the antibody comprises:

(i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 101and a light chain comprising the amino acid sequence of SEQ ID NO: 90;

(ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 97and a light chain comprising the amino acid sequence of SEQ ID NO: 85;

(iii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 98and a light chain comprising the amino acid sequence of SEQ ID NO: 85;

(iv) a heavy chain comprising the amino acid sequence of SEQ ID NO: 99and a light chain comprising the amino acid sequence of SEQ ID NO: 85;

(v) a heavy chain comprising the amino acid sequence of SEQ ID NO: 100and a light chain comprising the amino acid sequence of SEQ ID NO: 89;

(vi) a heavy chain comprising the amino acid sequence of SEQ ID NO: 100and a light chain comprising the amino acid sequence of SEQ ID NO: 90;

(vii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 101and a light chain comprising the amino acid sequence of SEQ ID NO: 89;

(viii) a heavy chain comprising the amino acid sequence of SEQ ID NO:102 and a light chain comprising the amino acid sequence of SEQ ID NO:93;

(ix) a heavy chain comprising the amino acid sequence of SEQ ID NO: 103and a light chain comprising the amino acid sequence of SEQ ID NO: 95;or

(x) a heavy chain comprising the amino acid sequence of SEQ ID NO: 102and a light chain comprising the amino acid sequence of SEQ ID NO: 95.

In some embodiments, the antibody does not specifically bind to thetransferrin binding site of the transferrin receptor and/or the antibodydoes not inhibit binding of transferrin to the transferrin receptor. Insome embodiments, the antibody is cross-reactive with extracellularepitopes of two or more of a human, non-human primate and rodenttransferrin receptor. In some embodiments, the complex is configured topromote transferrin receptor mediated internalization of the molecularpayload into a muscle cell.

In some embodiments, the molecular payload is an oligonucleotide. Insome embodiments, the oligonucleotide comprises at least 15 consecutivenucleotides of SEQ ID NOs: 148-383 and 621-638, wherein any one or moreof the thymidine bases (T's) in the oligonucleotide may optionally be auridine base (U) and/or any one or more of the U's may optionally be aT. In some embodiments, the oligonucleotide comprises a sequencecomprising any one of SEQ ID NOs: 159, 162, 172, 174, 180, 182, 188,190, 195, 196, 201, 203, 212, 215, 218, 222, 248, and 264, wherein anyone or more of the U's in the oligonucleotide may optionally be a T. Insome embodiments, the oligonucleotide comprises a region ofcomplementarity to at least 15 consecutive nucleotides of any one of SEQID NO: 384-619.

In some embodiments, the oligonucleotide mediates RNAse H-mediatedcleavage of a DMPK mRNA transcript.

In some embodiments, the oligonucleotide comprises a 5′-X—Y—Z-3′formula, wherein X and Z are flanking regions comprising one or more2′-modified nucleosides selected from the group consisting of:2′-O-methyl, 2′-fluoro, 2′-O-methoxyethyl, and 2′,4′-bridgednucleosides, and wherein Y is a gap region and each nucleoside in Y is a2′-deoxyribonucleoside.

In some embodiments, the oligonucleotide comprises one or morephosphorothioate internucleoside linkages.

In some embodiments, the antibody is covalently linked to the molecularpayload via a cleavable linker. In some embodiments, the cleavablelinker comprises a valine-citrulline sequence.

In some embodiments, the antibody is covalently linked to the molecularpayload via conjugation to a lysine residue or a cysteine residue of theantibody.

In some embodiments, reducing expression comprises reducing RNA levelsof DMPK, optionally wherein the reduced RNA levels are in the nucleus ofa cell, optionally wherein the cell is a muscle cell. In someembodiments, the DMPK is encoded from an allele comprising adisease-associated repeat.

Another aspect of the present disclosure relates to a method of reducingDMPK expression in a cell, the method comprising contacting the cellwith a complex disclosed herein in an effective amount for promotinginternalization of the molecular payload in the cell, optionally whereinthe cell is a muscle cell.

Another aspect of the present disclosure relates to a method of treatinga subject having an expansion of a disease-associated-repeat of a DMPKallele that is associated with myotonic dystrophy, the method comprisingadministering to the subject an effective amount of a complex disclosedherein. In some embodiments, the disease-associated-repeat comprisesrepeating units of a CTG trinucleotide sequence. In some embodiments,the complex is intravenously administered to the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a non-limiting schematic showing the effect oftransfecting Hepa 1-6 cells with an antisense oligonucleotide thattargets DMPK (ASO300) on expression levels of DMPK relative to a vehicletransfection.

FIG. 2A depicts a non-limiting schematic showing an HIL-HPLC traceobtained during purification of a muscle targeting complex comprising ananti-transferrin receptor antibody covalently linked to a DMPK antisenseoligonucleotide.

FIG. 2B depicts a non-limiting image of an SDS-PAGE analysis of a muscletargeting complex.

FIG. 3 depicts a non-limiting schematic showing the ability of a muscletargeting RI7 217 Fab antibody-oligonucleotide complex (DTX-C-008)comprising ASO300 to reduce expression levels of DMPK.

FIGS. 4A-4E depict non-limiting schematics showing the ability of amuscle targeting RI7 217 Fab antibody-oligonucleotide complex(DTX-C-008) comprising ASO300 to reduce expression levels of DMPK inmouse muscle tissues in vivo, relative to a vehicle treatment, treatmentwith naked ASO300, or treatment with a control non-targeting complex(DTX-C-007). (N=3 C57B1/6 WT mice)

FIGS. 5A-5B depict non-limiting schematics showing the tissueselectivity of a muscle targeting RI7 217 Fab antibody-oligonucleotidecomplex (DTX-C-008) comprising ASO300. The muscle targeting complex(DTX-C-008) comprising ASO300 does not reduce expression levels of DMPKin mouse brain or spleen tissues in vivo, relative to a vehicletreatment, treatment with naked ASO300, or treatment with a controlnon-targeting complex (DTX-C-007). (N=3 C57B1/6 WT mice).

FIGS. 6A-6F depict non-limiting schematics showing the ability of amuscle targeting RI7 217 Fab antibody-oligonucleotide complex(DTX-C-008) comprising ASO300 to reduce expression levels of DMPK inmouse muscle tissues in vivo, relative to a vehicle treatment, treatmentwith naked ASO300, or treatment with a control non-targeting complex(DTX-C-007). (N=5 C57B1/6 WT mice)

FIGS. 7A-7L depict non-limiting schematics showing the ability of amuscle targeting antibody-oligonucleotide complex (DTX-C-012) comprisingASO300 covalently linked to an anti-hTfR antibody to reduce expressionlevels of DMPK in cynomolgus monkey muscle tissues in vivo, relative toa vehicle treatment (saline) and compared to naked ASO300. (N=3 malecynomolgus monkeys)

FIGS. 8A-8B depict non-limiting schematics showing the ability of amuscle targeting antibody-oligonucleotide complex (DTX-C-012) comprisingASO300 covalently linked to an anti-hTfR antibody to reduce expressionlevels of DMPK in cynomolgus monkey smooth muscle tissues in vivo,relative to a vehicle treatment (saline) and compared to naked ASO300.(N=3 male cynomolgus monkeys)

FIGS. 9A-9D depict non-limiting schematics showing the tissueselectivity of a muscle targeting antibody-oligonucleotide complex(DTX-C-012) comprising ASO300 covalently linked to an anti-hTfRantibody. The muscle targeting complex comprising DMPK-ASO does notreduce expression levels of DMPK in cynomolgus monkey kidney, brain, orspleen tissues in vivo, relative to a vehicle treatment. (N=3 malecynomolgus monkeys)

FIG. 10 shows normalized DMPK mRNA tissue expression levels acrossseveral tissue types in cynomolgus monkeys. (N=3 male cynomolgusmonkeys)

FIGS. 11A-11B depict non-limiting schematics showing the ability of amuscle targeting RI7 217 Fab antibody-oligonucleotide complex(DTX-C-008) comprising ASO300 to reduce expression levels of DMPK inmouse muscle tissues in vivo for up to 28 days after dosing withDTX-C-008, relative to a vehicle treatment (saline) and compared tonaked ASO300.

FIG. 12 shows that a single dose of a muscle targeting complex(DTX-C-012) comprising ASO300 covalently linked to an anti-hTFR antibodyis safe and tolerated in cynomolgus monkeys. (N=3 male cynomolgusmonkeys)

FIGS. 13A-13B depict non-limiting schematics showing the ability of amuscle targeting RI7 217 Fab antibody-oligonucleotide complex(DTX-C-008) comprising ASO300 to reduce expression levels of DMPK inmouse muscle tissues in vivo for up to twelve weeks after dosing withDTX-C-008, relative to a vehicle treatment (PBS); and compared to acontrol IgG2a Fab antibody-oligonucleotide complex (DTX-C-007) and nakedDMPK ASO (ASO300). (N=5 C57B1/6 WT mice)

FIGS. 14A-14B depict non-limiting schematics showing the ability of amuscle-targeting RI7 217 Fab antibody-oligonucleotide complex(DTX-C-008) comprising ASO300 to target nuclear mutant DMPK RNA in amouse model. (N=6 mice)

FIGS. 15A-15B depict non-limiting schematics showing the ability of amuscle-targeting RI7 217 Fab antibody-ASO complex (DTX-Actin) comprisingan oligonucleotide that targets actin to dose-dependently reduceexpression levels of actin and functional grades of myotonia in muscletissues. (N=2 HSA^(LR) mice)

FIGS. 16A-16C depict non-limiting schematics showing that amuscle-targeting RI7 217 Fab antibody-oligonucleotide complex(DTX-C-008) comprising ASO300 is capable of significantly reducing theprolonged QTc interval in a mouse model for validation of the functionalcorrection of arrhythmia in a DM1 cardiac model. (N=10 mice)

FIGS. 17A-17B depict non-limiting schematics showing that amuscle-targeting antibody-oligonucleotide complex (DTX-C-012) comprisingASO300 antisense oligonucleotide covalently linked to an anti-hTfRantibody is capable of reducing expression levels of DMPK and correctingsplicing of a DMPK-specific target gene (Bin1) in human cells from a DM1patient. (N=3)

FIGS. 18A-18C depict non-limiting schematics showing the dose responseof selected antisense oligonucleotides in DMPK knockdown in human DM1myotubes. ASO300 was used as control. All tested oligonucleotides showedactivity in DMPK knockdown. Statistical analysis: One-way ANOVA withTukey's HSD post-hoc test vs. naked ASO300 treatment; *p<0.05, **p<0.01,***p<0.001, ****p<0.0001.

FIGS. 19A-19B depict non-limiting schematics showing the dose responseof selected antisense oligonucleotides in DMPK knockdown in non-humanprimate (NHP) DM1 myotubes. ASO300 was used as control. All testedoligonucleotides showed activity in DMPK knockdown.

FIG. 20 shows the serum stability of the linker used for linking ananti-TfR antibody and a molecular payload (e.g., an oligonucleotide) invarious species over time after intravenous administration.

FIGS. 21A-21F show binding of humanized anti-TfR Fabs to human TfR1(hTfR1) or cynomolgus monkey TfR1 (cTfR1), as measured by ELISA. FIG.21A shows binding of humanized 3M12 variants to hTfR1. FIG. 21B showsbinding of humanized 3M12 variants to cTfR1. FIG. 21C shows binding ofhumanized 3A4 variants to hTfR1. FIG. 21D shows binding of humanized 3A4variants to cTfR1. FIG. 21E shows binding of humanized 5H12 variants tohTfR1. FIG. 21F shows binding of humanized 5H12 variants to hTfR1.

FIG. 22 shows the quantified cellular uptake of anti-TfR Fab conjugatesinto rhabdomyosarcoma (RD) cells. The molecular payload in the testedconjugates are DMPK-targeting oligonucleotides and the uptake of theconjugates were facilitated by indicated anti-TfR Fabs. Conjugateshaving a negative control Fab (anti-mouse TfR) or a positive control Fab(anti-human TfR1) are also included this assay. Cells were incubatedwith indicated conjugate at a concentration of 100 nM for 4 hours.Cellular uptake was measured by mean Cypher5e fluorescence.

FIGS. 23A-23F show binding of oligonucleotide-conjugated or unconjugatedhumanized anti-TfR Fabs to human TfR1 (hTfR1) and cynomolgus monkey TfR1(cTfR1), as measured by ELISA. FIG. 23A shows the binding of humanized3M12 variants alone or in conjugates with a DMPK targeting oligo tohTfR1. FIG. 23B shows the binding of humanized 3M12 variants alone or inconjugates with a DMPK targeting oligo to cTfR1. FIG. 23C shows thebinding of humanized 3A4 variants alone or in conjugates with a DMPKtargeting oligo to hTfR1. FIG. 23D shows the binding of humanized 3A4variants alone or in conjugates with a DMPK targeting oligo to cTfR1.FIG. 23E shows the binding of humanized 5H12 variants alone or inconjugates with a DMPK targeting oligo to hTfR1. FIG. 23F shows thebinding of humanized 5H12 variants alone or in conjugates with a DMPKtargeting oligo to cTfR1. The respective EC₅₀ values are also shown.

FIG. 24 shows DMPK expression in RD cells treated with DMPK-targetingoligonucleotides relative to cells treated with PBS. The durationtreatment was 3 days. DMPK-targeting oligonucleotides were delivered tothe cells as free oligonucleotides (gymnotic uptake, “free”) or withtransfection reagent (“trans”).

FIG. 25 shows DMPK expression in RD cells treated with variousconcentrations of conjugates containing the indicated humanized anti-TfRantibodies conjugated to a DMPK-targeting antisense oligonucleotide(ASO300). The duration of treatment was 3 days. ASO300 delivered usingtransfection agents (labeled “Trans”) was used as control.

FIG. 26 shows results of splicing correction in Atp2a1 by an anti-TfR1antibody-oligonucleotide conjugate (Ab-ASO) in the HSA-LR mouse model ofDM1, measured in the gastrocnemius muscle. The anti-TfR antibody used isRI7 217 Fab and the oligonucleotide is targeting skeletal actin.

FIG. 27 shows splicing correction in more than 30 different RNAs relatedto DM1, measured in the gastrocnemius muscle of HSA-LR mice treatedanti-TfR1 antibody-oligonucleotide (Ab-ASO) conjugate or saline. Theanti-TfR antibody used is RI7 217 Fab and the oligonucleotide istargeting human skeletal actin.

FIG. 28 shows splicing derangement in quadriceps, gastrocnemius, ortibialis anterior muscles of HSA-LR mice treated with anti-TfR1antibody-oligonucleotide conjugate (Ab-ASO) or saline. The datarepresent composite splicing derangement measured in the more than 30RNAs shown in FIG. 27 .

FIG. 29 shows myotonia grade measured in quadriceps, gastrocnemius, andtibialis anterior muscles of HSA-LR mice treated with saline,unconjugated oligonucleotide (ASO), or anti-TfR1antibody-oligonucleotide conjugate (Ab-ASO). Myotonia was measured byelectromyography (EMG), and graded 0, 1, 2, or 3 based on the frequencyof myotonic discharge.

FIGS. 30A-30E show in vivo activity of conjugates containing designatedanti-TfR Fabs (control, 3M12 VH3/VK2, 3M12 VH4/VK3, and 3A4 VH3N54S/VK4) conjugated to DMPK-targeting oligonucleotide in reducing DMPKmRNA expression in mice expressing human TfR1 (hTfR1 knock-in mice).FIG. 30A shows the experimental design (e.g., IV dosage, dosingfrequency). DMPK mRNA levels were measured 14 days post first dose inthe tibialis anterior (FIG. 30B), gastrocnemius (FIG. 30C), heart (FIG.30D), and diaphragm (FIG. 30E), of the mice.

FIGS. 31A-31C show that conjugates containing anti-TfR antibodyconjugated to DMPK-targeting oligonucleotide corrected splicing andreduced foci in CM-DM1-32F primary cells expressing a DMPK mutant mRNAcontaining 380 CUG repeats. FIG. 31A shows that the conjugates reducedmutant DMPK mRNA expression. FIG. 31B shows that the conjugatescorrected BIN1 Exon 11 splicing. FIG. 31C shows images of a fluorescencein situ hybridization (FISH) analysis and quantification of the images,demonstrating that the conjugated reduced nuclear foci formed by themutant DMPK mRNA. In the microscopy images shown in the top panels ofFIG. 31C, the light rounded shapes show cell nuclei, and the brightpuncta within the nuclei of the DM1 cells (right three microscopypanels) show CUG foci.

FIG. 32 shows ELISA measurements of binding of anti-TfR Fab 3M12 VH4/Vk3to recombinant human (circles), cynomolgus monkey (squares), mouse(upward triangles), or rat (downward triangles) TfR1 protein, at a rangeof concentrations from 230 pM to 500 nM of the Fab. Measurement resultsshow that the anti-TfR Fab is reactive with human and cynomolgus monkeyTfR1. Binding was not observed to mouse or rat recombinant TfR1. Data isshown as relative fluorescent units normalized to baseline.

FIG. 33 shows results of an ELISA testing the affinity of anti-TfR Fab3M12 VH4/Vk3 to recombinant human TfR1 or TfR2 over a range ofconcentrations from 230 pM to 500 nM of Fab. The data are presented asrelative fluorescence units normalized to baseline. The resultsdemonstrate that the Fab does not bind recombinant human TfR2.

FIG. 34 shows the serum stability of the linker used for linkinganti-TfR Fab 3M12 VH4/Vk3 to a control antisense oligonucleotide over 72hours incubation in PBS or in rat, mouse, cynomolgus monkey or humanserum.

FIGS. 35A-35B show splicing correction in more than 30 different RNAsknown to be mis-spliced in DM1 patients, measured in the tibialisanterior (FIG. 35A) or the quadriceps (FIG. 35B) of HSA-LR mice treatedwith a single dose of anti-TfR antibody-oligonucleotide (Ab-ASO)conjugate or saline. The anti-TfR antibody used is RI7 217 Fab and theoligonucleotide targets skeletal actin (ACTA1).

FIGS. 36A-36C show EMG myotonia grade in quadriceps (FIG. 36A),gastrocnemius (FIG. 36B), and tibialis anterior (FIG. 36C) of HSA-LRmice treated with vehicle, a single dose of unconjugated ASO, or asingle dose of anti-TfR antibody-ASO conjugate (Ab-ASO). The anti-TfRantibody used is RI7 217 Fab and the oligonucleotide targets humanskeletal actin (ACTA1).

FIG. 37 shows human ACTA1 expression measured by qPCR in HSA^(LR) DM1mice after a single dose of naked ASO or dose equivalent of anti-TFRantibody-ASO conjugate (Ab-ASO), relative to vehicle-treated mice. Theanti-TfR antibody used is RI7 217 Fab and the oligonucleotide targetshuman skeletal actin (ACTA1).

FIGS. 38A-38C show ACTA1 expression in quadriceps (FIG. 38A),gastrocnemius (FIG. 38B), and tibialis anterior (FIG. 38C) in HSA^(LR)DM1 mice after a single dose of 10 mg/kg naked ASO, 20 mg/kg naked ASO,or dose equivalents of anti-TFR antibody-ASO conjugate (Ab-ASO),relative to vehicle-treated mice. The anti-TfR antibody used is RI7 217Fab and the oligonucleotide targets human skeletal actin (ACTA1).(*p<0.05; ***p<0.001)

DETAILED DESCRIPTION OF INVENTION

Aspects of the disclosure relate to a recognition that while certainmolecular payloads (e.g., oligonucleotides, peptides, small molecules)can have beneficial effects in muscle cells, it has proven challengingto effectively target such cells. As described herein, the presentdisclosure provides complexes comprising muscle-targeting agentscovalently linked to molecular payloads in order to overcome suchchallenges. In some embodiments, the complexes are particularly usefulfor delivering molecular payloads that inhibit the expression oractivity of target genes in muscle cells, e.g., in a subject having orsuspected of having a rare muscle disease. For example, in someembodiments, complexes are provided for targeting a DMPK allele thatcomprises an expanded disease-associated-repeat to treat subjects havingDM1. In some embodiments, complexes provided herein may compriseoligonucleotides that inhibit expression of a DMPK allele comprising anexpanded disease-associated-repeat. As another example, complexes maycomprise oligonucleotides that interfere with the binding of adisease-associated DMPK mRNA to a muscleblind-like protein (e.g., MBNL1,2, and/or (e.g., and) 3), thereby reducing a toxic effect of adisease-associated DMPK allele. In some embodiments, synthetic nucleicacid payloads (e.g., DNA or RNA payloads) may be used that express oneor more proteins that reduce a toxic effect of a disease-associated DMPKallele. In some embodiments, complexes may comprise molecular payloadsof synthetic cDNAs and/or (e.g., and) synthetic mRNAs, e.g., thatexpress one or more muscleblind-like-proteins (e.g., MBNL1, 2, and/or(e.g., and) 3) or fragments thereof. In some embodiments, complexes maycomprise molecular payloads such as guide molecules (e.g., guide RNAs)that are capable of targeting nucleic acid programmable nucleases (e.g.,Cas9) to a sequence at or near a disease-associated repeat sequence ofDMPK. In some embodiments, such nucleic programmable nucleases could beused to cleave part or all of a disease-associated repeat sequence froma DMPK gene.

Further aspects of the disclosure, including a description of definedterms, are provided below.

I. Definitions

Administering: As used herein, the terms “administering” or“administration” means to provide a complex to a subject in a mannerthat is physiologically and/or (e.g., and) pharmacologically useful(e.g., to treat a condition in the subject).

Approximately: As used herein, the term “approximately” or “about,” asapplied to one or more values of interest, refers to a value that issimilar to a stated reference value. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, orless in either direction (greater than or less than) of the statedreference value unless otherwise stated or otherwise evident from thecontext (except where such number would exceed 100% of a possiblevalue).

Antibody: As used herein, the term “antibody” refers to a polypeptidethat includes at least one immunoglobulin variable domain or at leastone antigenic determinant, e.g., paratope that specifically binds to anantigen. In some embodiments, an antibody is a full-length antibody. Insome embodiments, an antibody is a chimeric antibody. In someembodiments, an antibody is a humanized antibody. However, in someembodiments, an antibody is a Fab fragment, a Fab′, a F(ab′)2 fragment,a Fv fragment or a scFv fragment. In some embodiments, an antibody is ananobody derived from a camelid antibody or a nanobody derived fromshark antibody. In some embodiments, an antibody is a diabody. In someembodiments, an antibody comprises a framework having a human germlinesequence. In another embodiment, an antibody comprises a heavy chainconstant domain selected from the group consisting of IgG, IgG1, IgG2,IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE constantdomains. In some embodiments, an antibody comprises a heavy (H) chainvariable region (abbreviated herein as VH), and/or (e.g., and) a light(L) chain variable region (abbreviated herein as VL). In someembodiments, an antibody comprises a constant domain, e.g., an Fcregion. An immunoglobulin constant domain refers to a heavy or lightchain constant domain. Human IgG heavy chain and light chain constantdomain amino acid sequences and their functional variations are known.With respect to the heavy chain, in some embodiments, the heavy chain ofan antibody described herein can be an alpha (α), delta (Δ), epsilon(ε), gamma (γ) or mu (μ) heavy chain. In some embodiments, the heavychain of an antibody described herein can comprise a human alpha (α),delta (Δ), epsilon (ε), gamma (γ) or mu (μ) heavy chain. In a particularembodiment, an antibody described herein comprises a human gamma 1 CH1,CH2, and/or (e.g., and) CH3 domain. In some embodiments, the amino acidsequence of the VH domain comprises the amino acid sequence of a humangamma (γ) heavy chain constant region, such as any known in the art.Non-limiting examples of human constant region sequences have beendescribed in the art, e.g., see U.S. Pat. No. 5,693,780 and Kabat E A etal., (1991) supra. In some embodiments, the VH domain comprises an aminoacid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or atleast 99% identical to any of the variable chain constant regionsprovided herein. In some embodiments, an antibody is modified, e.g.,modified via glycosylation, phosphorylation, sumoylation, and/or (e.g.,and) methylation. In some embodiments, an antibody is a glycosylatedantibody, which is conjugated to one or more sugar or carbohydratemolecules. In some embodiments, the one or more sugar or carbohydratemolecule are conjugated to the antibody via N-glycosylation,O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment),and/or (e.g., and) phosphoglycosylation. In some embodiments, the one ormore sugar or carbohydrate molecule are monosaccharides, disaccharides,oligosaccharides, or glycans. In some embodiments, the one or more sugaror carbohydrate molecule is a branched oligosaccharide or a branchedglycan. In some embodiments, the one or more sugar or carbohydratemolecule includes a mannose unit, a glucose unit, an N-acetylglucosamineunit, an N-acetylgalactosamine unit, a galactose unit, a fucose unit, ora phospholipid unit. In some embodiments, an antibody is a constructthat comprises a polypeptide comprising one or more antigen bindingfragments of the disclosure linked to a linker polypeptide or animmunoglobulin constant domain. Linker polypeptides comprise two or moreamino acid residues joined by peptide bonds and are used to link one ormore antigen binding portions. Examples of linker polypeptides have beenreported (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci.USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123).Still further, an antibody may be part of a larger immunoadhesionmolecule, formed by covalent or noncovalent association of the antibodyor antibody portion with one or more other proteins or peptides.Examples of such immunoadhesion molecules include use of thestreptavidin core region to make a tetrameric scFv molecule (Kipriyanov,S. M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and useof a cysteine residue, a marker peptide and a C-terminal polyhistidinetag to make bivalent and biotinylated scFv molecules (Kipriyanov, S. M.,et al. (1994) Mol. Immunol. 31:1047-1058).

CDR: As used herein, the term “CDR” refers to the complementaritydetermining region within antibody variable sequences. A typicalantibody molecule comprises a heavy chain variable region (VH) and alight chain variable region (VL), which are usually involved in antigenbinding. The VH and VL regions can be further subdivided into regions ofhypervariability, also known as “complementarity determining regions”(“CDR”), interspersed with regions that are more conserved, which areknown as “framework regions” (“FR”). Each VH and VL is typicallycomposed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The extent of the framework region and CDRs can be preciselyidentified using methodology known in the art, for example, by the Kabatdefinition, the IMGT definition, the Chothia definition, the AbMdefinition, and/or (e.g., and) the contact definition, all of which arewell known in the art. See, e.g., Kabat, E. A., et al. (1991) Sequencesof Proteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242; IMGT®, theInternational ImMunoGeneTics Information System® http://www.imgt.org,Lefranc, M.-P. et al., Nucleic Acids Res., 27:209-212 (1999); Ruiz, M.et al., Nucleic Acids Res., 28:219-221 (2000); Lefranc, M.-P., NucleicAcids Res., 29:207-209 (2001); Lefranc, M.-P., Nucleic Acids Res.,31:307-310 (2003); Lefranc, M.-P. et al., In Silico Biol., 5, 0006(2004) [Epub], 5:45-60 (2005); Lefranc, M.-P. et al., Nucleic AcidsRes., 33:D593-597 (2005); Lefranc, M.-P. et al., Nucleic Acids Res.,37:D1006-1012 (2009); Lefranc, M.-P. et al., Nucleic Acids Res.,43:D413-422 (2015); Chothia et al., (1989) Nature 342:877; Chothia, C.et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al (1997) J.Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143(2004). See also hgmp.mrc.ac.uk and bioinf.org.uk/abs. As used herein, aCDR may refer to the CDR defined by any method known in the art. Twoantibodies having the same CDR means that the two antibodies have thesame amino acid sequence of that CDR as determined by the same method,for example, the IMGT definition.

There are three CDRs in each of the variable regions of the heavy chainand the light chain, which are designated CDR1, CDR2 and CDR3, for eachof the variable regions. The term “CDR set” as used herein refers to agroup of three CDRs that occur in a single variable region capable ofbinding the antigen. The exact boundaries of these CDRs have beendefined differently according to different systems. The system describedby Kabat (Kabat et al., Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987) and (1991)) notonly provides an unambiguous residue numbering system applicable to anyvariable region of an antibody, but also provides precise residueboundaries defining the three CDRs. These CDRs may be referred to asKabat CDRs. Sub-portions of CDRs may be designated as L1, L2 and L3 orH1, H2 and H3 where the “L” and the “H” designates the light chain andthe heavy chains regions, respectively. These regions may be referred toas Chothia CDRs, which have boundaries that overlap with Kabat CDRs.Other boundaries defining CDRs overlapping with the Kabat CDRs have beendescribed by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J MolBiol 262(5):732-45 (1996)). Still other CDR boundary definitions may notstrictly follow one of the above systems, but will nonetheless overlapwith the Kabat CDRs, although they may be shortened or lengthened inlight of prediction or experimental findings that particular residues orgroups of residues or even entire CDRs do not significantly impactantigen binding. The methods used herein may utilize CDRs definedaccording to any of these systems. Examples of CDR definition systemsare provided in Table 1.

TABLE 1 CDR Definitions IMGT¹ Kabat² Chothia³ CDR-H1 27-38 31-35 26-32CDR-H2 56-65 50-65 53-55 CDR-H3 105-116/117  95-102  96-101 CDR-L1 27-3824-34 26-32 CDR-L2 56-65 50-56 50-52 CDR-L3 105-116/117 89-97 91-96¹IMGT ®, the international ImMunoGeneTics information system ®,imgt.org, Lefranc, M.-P. et al., Nucleic Acids Res., 27: 209-212 (1999)²Kabat et al. (1991) Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242 ³Chothia et al., J. Mol. Biol. 196: 901-917(1987))

CDR-grafted antibody: The term “CDR-grafted antibody” refers toantibodies which comprise heavy and light chain variable regionsequences from one species but in which the sequences of one or more ofthe CDR regions of VH and/or (e.g., and) VL are replaced with CDRsequences of another species, such as antibodies having murine heavy andlight chain variable regions in which one or more of the murine CDRs(e.g., CDR3) has been replaced with human CDR sequences.

Chimeric antibody: The term “chimeric antibody” refers to antibodieswhich comprise heavy and light chain variable region sequences from onespecies and constant region sequences from another species, such asantibodies having murine heavy and light chain variable regions linkedto human constant regions.

Complementary: As used herein, the term “complementary” refers to thecapacity for precise pairing between two nucleotides or two sets ofnucleotides. In particular, complementary is a term that characterizesan extent of hydrogen bond pairing that brings about binding between twonucleotides or two sets of nucleotides. For example, if a base at oneposition of an oligonucleotide is capable of hydrogen bonding with abase at the corresponding position of a target nucleic acid (e.g., anmRNA), then the bases are considered to be complementary to each otherat that position. Base pairings may include both canonical Watson-Crickbase pairing and non-Watson-Crick base pairing (e.g., Wobble basepairing and Hoogsteen base pairing). For example, in some embodiments,for complementary base pairings, adenosine-type bases (A) arecomplementary to thymidine-type bases (T) or uracil-type bases (U), thatcytosine-type bases (C) are complementary to guanosine-type bases (G),and that universal bases such as 3-nitropyrrole or 5-nitroindole canhybridize to and are considered complementary to any A, C, U, or T.Inosine (I) has also been considered in the art to be a universal baseand is considered complementary to any A, C, U or T.

Conservative amino acid substitution: As used herein, a “conservativeamino acid substitution” refers to an amino acid substitution that doesnot alter the relative charge or size characteristics of the protein inwhich the amino acid substitution is made. Variants can be preparedaccording to methods for altering polypeptide sequence known to one ofordinary skill in the art such as are found in references which compilesuch methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook,et al., eds., Fourth Edition, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 2012, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York.Conservative substitutions of amino acids include substitutions madeamongst amino acids within the following groups: (a) M, I, L, V; (b) F,Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

Covalently linked: As used herein, the term “covalently linked” refersto a characteristic of two or more molecules being linked together viaat least one covalent bond. In some embodiments, two molecules can becovalently linked together by a single bond, e.g., a disulfide bond ordisulfide bridge, that serves as a linker between the molecules.However, in some embodiments, two or more molecules can be covalentlylinked together via a molecule that serves as a linker that joins thetwo or more molecules together through multiple covalent bonds. In someembodiments, a linker may be a cleavable linker. However, in someembodiments, a linker may be a non-cleavable linker.

Cross-reactive: As used herein and in the context of a targeting agent(e.g., antibody), the term “cross-reactive,” refers to a property of theagent being capable of specifically binding to more than one antigen ofa similar type or class (e.g., antigens of multiple homologs, paralogs,or orthologs) with similar affinity or avidity. For example, in someembodiments, an antibody that is cross-reactive against human andnon-human primate antigens of a similar type or class (e.g., a humantransferrin receptor and non-human primate transferrin receptor) iscapable of binding to the human antigen and non-human primate antigenswith a similar affinity or avidity. In some embodiments, an antibody iscross-reactive against a human antigen and a rodent antigen of a similartype or class. In some embodiments, an antibody is cross-reactiveagainst a rodent antigen and a non-human primate antigen of a similartype or class. In some embodiments, an antibody is cross-reactiveagainst a human antigen, a non-human primate antigen, and a rodentantigen of a similar type or class.

Disease-associated-repeat: As used herein, the term“disease-associated-repeat” refers to a repeated nucleotide sequence ata genomic location for which the number of units of the repeatednucleotide sequence is correlated with and/or (e.g., and) directly orindirectly contributes to, or causes, genetic disease. Each repeatingunit of a disease associated repeat may be 2, 3, 4, 5 or morenucleotides in length. For example, in some embodiments, a diseaseassociated repeat is a dinucleotide repeat. In some embodiments, adisease associated repeat is a trinucleotide repeat. In someembodiments, a disease associated repeat is a tetranucleotide repeat. Insome embodiments, a disease associated repeat is a pentanucleotiderepeat. In some embodiments, embodiments, the disease-associated-repeatcomprises CAG repeats, CTG repeats, CUG repeats, CGG repeats, CCTGrepeats, or a nucleotide complement of any thereof. In some embodiments,a disease-associated-repeat is in a non-coding portion of a gene.However, in some embodiments, a disease-associated-repeat is in a codingregion of a gene. In some embodiments, a disease-associated-repeat isexpanded from a normal state to a length that directly or indirectlycontributes to, or causes, genetic disease. In some embodiments, adisease-associated-repeat is in RNA (e.g., an RNA transcript). In someembodiments, a disease-associated-repeat is in DNA (e.g., a chromosome,a plasmid). In some embodiments, a disease-associated-repeat is expandedin a chromosome of a germline cell. In some embodiments, adisease-associated-repeat is expanded in a chromosome of a somatic cell.In some embodiments, a disease-associated-repeat is expanded to a numberof repeating units that is associated with congenital onset of disease.In some embodiments, a disease-associated-repeat is expanded to a numberof repeating units that is associated with childhood onset of disease.In some embodiments, a disease-associated-repeat is expanded to a numberof repeating units that is associated with adult onset of disease.

DMPK: As used herein, the term “DMPK” refers to a gene that encodesmyotonin-protein kinase (also known as myotonic dystrophy protein kinaseor dystrophia myotonica protein kinase), a serine/threonine proteinkinase. Substrates for this enzyme may include myogenin, thebeta-subunit of the L-type calcium channels, and phospholemman. In someembodiments, DMPK may be a human (Gene ID: 1760), non-human primate(e.g., Gene ID: 456139, Gene ID: 715328), or rodent gene (e.g., Gene ID:13400). In humans, a CTG repeat expansion in the 3′ non-coding,untranslated region of DMPK is associated with myotonic dystrophy type I(DM1). In addition, multiple human transcript variants (e.g., asannotated under GenBank RefSeq Accession Numbers: NM_001081563.2,NM_004409.4, NM_001081560.2, NM_001081562.2, NM_001288764.1,NM_001288765.1, and NM_001288766.1) have been characterized that encodedifferent protein isoforms.

DMPK allele: As used herein, the term “DMPK allele” refers to any one ofalternative forms (e.g., wild-type or mutant forms) of a DMPK gene. Insome embodiments, a DMPK allele may encode for wild-typemyotonin-protein kinase that retains its normal and typical functions.In some embodiments, a DMPK allele may comprise one or moredisease-associated-repeat expansions. In some embodiments, normalsubjects have two DMPK alleles comprising in the range of 5 to 37 repeatunits. In some embodiments, the number of CTG repeat units in subjectshaving DM1 is in the range of ˜50 to ˜3,000+ with higher numbers ofrepeats leading to an increased severity of disease. In someembodiments, mildly affected DM1 subjects have at least one DMPK allelehaving in the range of 50 to 150 repeat units. In some embodiments,subjects with classic DM1 have at least one DMPK allele having in therange of 100 to 1,000 or more repeat units. In some embodiments,subjects having DM1 with congenital onset may have at least one DMPKallele comprising more than 2,000 repeat units.

Framework: As used herein, the term “framework” or “framework sequence”refers to the remaining sequences of a variable region minus the CDRs.Because the exact definition of a CDR sequence can be determined bydifferent systems, the meaning of a framework sequence is subject tocorrespondingly different interpretations. The six CDRs (CDR-L1, CDR-L2,and CDR-L3 of light chain and CDR-H1, CDR-H2, and CDR-H3 of heavy chain)also divide the framework regions on the light chain and the heavy chaininto four sub-regions (FR1, FR2, FR3 and FR4) on each chain, in whichCDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, andCDR3 between FR3 and FR4. Without specifying the particular sub-regionsas FR1, FR2, FR3 or FR4, a framework region, as referred by others,represents the combined FRs within the variable region of a single,naturally occurring immunoglobulin chain. As used herein, a FRrepresents one of the four sub-regions, and FRs represents two or moreof the four sub-regions constituting a framework region. Human heavychain and light chain acceptor sequences are known in the art. In oneembodiment, the acceptor sequences known in the art may be used in theantibodies disclosed herein.

Human antibody: The term “human antibody”, as used herein, is intendedto include antibodies having variable and constant regions derived fromhuman germline immunoglobulin sequences. The human antibodies of thedisclosure may include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

Humanized antibody: The term “humanized antibody” refers to antibodieswhich comprise heavy and light chain variable region sequences from anon-human species (e.g., a mouse) but in which at least a portion of theVH and/or (e.g., and) VL sequence has been altered to be more“human-like”, i.e., more similar to human germline variable sequences.One type of humanized antibody is a CDR-grafted antibody, in which humanCDR sequences are introduced into non-human VH and VL sequences toreplace the corresponding nonhuman CDR sequences. In one embodiment,humanized anti-transferrin receptor antibodies and antigen bindingportions are provided. Such antibodies may be generated by obtainingmurine anti-transferrin receptor monoclonal antibodies using traditionalhybridoma technology followed by humanization using in vitro geneticengineering, such as those disclosed in Kasaian et al PCT publicationNo. WO 2005/123126 A2.

Internalizing cell surface receptor: As used herein, the term,“internalizing cell surface receptor” refers to a cell surface receptorthat is internalized by cells, e.g., upon external stimulation, e.g.,ligand binding to the receptor. In some embodiments, an internalizingcell surface receptor is internalized by endocytosis. In someembodiments, an internalizing cell surface receptor is internalized byclathrin-mediated endocytosis. However, in some embodiments, aninternalizing cell surface receptor is internalized by aclathrin-independent pathway, such as, for example, phagocytosis,macropinocytosis, caveolae- and raft-mediated uptake or constitutiveclathrin-independent endocytosis. In some embodiments, the internalizingcell surface receptor comprises an intracellular domain, a transmembranedomain, and/or (e.g., and) an extracellular domain, which may optionallyfurther comprise a ligand-binding domain. In some embodiments, a cellsurface receptor becomes internalized by a cell after ligand binding. Insome embodiments, a ligand may be a muscle-targeting agent or amuscle-targeting antibody. In some embodiments, an internalizing cellsurface receptor is a transferrin receptor.

Isolated antibody: An “isolated antibody”, as used herein, is intendedto refer to an antibody that is substantially free of other antibodieshaving different antigenic specificities (e.g., an isolated antibodythat specifically binds transferrin receptor is substantially free ofantibodies that specifically bind antigens other than transferrinreceptor). An isolated antibody that specifically binds transferrinreceptor complex may, however, have cross-reactivity to other antigens,such as transferrin receptor molecules from other species. Moreover, anisolated antibody may be substantially free of other cellular materialand/or (e.g., and) chemicals.

Kabat numbering: The terms “Kabat numbering”, “Kabat definitions and“Kabat labeling” are used interchangeably herein. These terms, which arerecognized in the art, refer to a system of numbering amino acidresidues which are more variable (i.e. hypervariable) than other aminoacid residues in the heavy and light chain variable regions of anantibody, or an antigen binding portion thereof (Kabat et al. (1971)Ann. NY Acad, Sci. 190:382-391 and, Kabat, E. A., et al. (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242).For the heavy chain variable region, the hypervariable region rangesfrom amino acid positions 31 to 35 for CDR1, amino acid positions 50 to65 for CDR2, and amino acid positions 95 to 102 for CDR3. For the lightchain variable region, the hypervariable region ranges from amino acidpositions 24 to 34 for CDR1, amino acid positions 50 to 56 for CDR2, andamino acid positions 89 to 97 for CDR3.

Molecular payload: As used herein, the term “molecular payload” refersto a molecule or species that functions to modulate a biologicaloutcome. In some embodiments, a molecular payload is linked to, orotherwise associated with a muscle-targeting agent. In some embodiments,the molecular payload is a small molecule, a protein, a peptide, anucleic acid, or an oligonucleotide. In some embodiments, the molecularpayload functions to modulate the transcription of a DNA sequence, tomodulate the expression of a protein, or to modulate the activity of aprotein. In some embodiments, the molecular payload is anoligonucleotide that comprises a strand having a region ofcomplementarity to a target gene.

Muscle-targeting agent: As used herein, the term, “muscle-targetingagent,” refers to a molecule that specifically binds to an antigenexpressed on muscle cells. The antigen in or on muscle cells may be amembrane protein, for example an integral membrane protein or aperipheral membrane protein. Typically, a muscle-targeting agentspecifically binds to an antigen on muscle cells that facilitatesinternalization of the muscle-targeting agent (and any associatedmolecular payload) into the muscle cells. In some embodiments, amuscle-targeting agent specifically binds to an internalizing, cellsurface receptor on muscles and is capable of being internalized intomuscle cells through receptor mediated internalization. In someembodiments, the muscle-targeting agent is a small molecule, a protein,a peptide, a nucleic acid (e.g., an aptamer), or an antibody. In someembodiments, the muscle-targeting agent is linked to a molecularpayload.

Muscle-targeting antibody: As used herein, the term, “muscle-targetingantibody,” refers to a muscle-targeting agent that is an antibody thatspecifically binds to an antigen found in or on muscle cells. In someembodiments, a muscle-targeting antibody specifically binds to anantigen on muscle cells that facilitates internalization of themuscle-targeting antibody (and any associated molecular payment) intothe muscle cells. In some embodiments, the muscle-targeting antibodyspecifically binds to an internalizing, cell surface receptor present onmuscle cells. In some embodiments, the muscle-targeting antibody is anantibody that specifically binds to a transferrin receptor.

Myotonic dystrophy (DM): As used herein, the term “Myotonic dystrophy(DM)” refers to a genetic disease caused by mutations in the DMPK geneor CNBP (ZNF9) gene that is characterized by muscle loss, muscleweakening, and muscle function. Two types of the disease, myotonicdystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2), have beendescribed. DM1 is associated with an expansion of a CTG trinucleotiderepeat in the 3′ non-coding region of DMPK. DM2 is associated with anexpansion of a CCTG tetranucleotide repeat in the first intron of ZNF9.In both DM1 and DM2, the nucleotide expansions lead to toxic RNA repeatscapable of forming hairpin structures that bind critical intracellularproteins, e.g., muscleblind-like proteins, with high affinity. Myotonicdystrophy, the genetic basis for the disease, and related symptoms aredescribed in the art (see, e.g. Thornton, C. A., “Myotonic Dystrophy”Neurol Clin. (2014), 32(3): 705-719.; and Konieczny et al. “Myotonicdystrophy: candidate small molecule therapeutics” Drug Discovery Today(2017), 22:11.) In some embodiments, subjects are born with a variationof DM1 called congenital myotonic dystrophy. Symptoms of congenitalmyotonic dystrophy are present from birth and include weakness of allmuscles, breathing problems, clubfeet, developmental delays andintellectual disabilities. DM1 is associated with Online MendelianInheritance in Man (OMIM) Entry #160900. DM2 is associated with OMIMEntry #602668.

Oligonucleotide: As used herein, the term “oligonucleotide” refers to anoligomeric nucleic acid compound of up to 200 nucleotides in length.Examples of oligonucleotides include, but are not limited to, RNAioligonucleotides (e.g., siRNAs, shRNAs), microRNAs, gapmers, mixmers,phosphorodiamidite morpholinos, peptide nucleic acids, aptamers, guidenucleic acids (e.g., Cas9 guide RNAs), etc. Oligonucleotides may besingle-stranded or double-stranded. In some embodiments, anoligonucleotide may comprise one or more modified nucleotides (e.g.2′-O-methyl sugar modifications, purine or pyrimidine modifications). Insome embodiments, an oligonucleotide may comprise one or more modifiedinternucleotide linkage. In some embodiments, an oligonucleotide maycomprise one or more phosphorothioate linkages, which may be in the Rpor Sp stereochemical conformation.

Recombinant antibody: The term “recombinant human antibody”, as usedherein, is intended to include all human antibodies that are prepared,expressed, created or isolated by recombinant means, such as antibodiesexpressed using a recombinant expression vector transfected into a hostcell (described in more details in this disclosure), antibodies isolatedfrom a recombinant, combinatorial human antibody library (Hoogenboom H.R., (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith W. E., (2002)Clin. Biochem. 35:425-445; Gavilondo J. V., and Larrick J. W. (2002)BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000) ImmunologyToday 21:371-378), antibodies isolated from an animal (e.g., a mouse)that is transgenic for human immunoglobulin genes (see e.g., Taylor, L.D., et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann S-A., andGreen L. L. (2002) Current Opinion in Biotechnology 13:593-597; LittleM. et al (2000) Immunology Today 21:364-370) or antibodies prepared,expressed, created or isolated by any other means that involves splicingof human immunoglobulin gene sequences to other DNA sequences. Suchrecombinant human antibodies have variable and constant regions derivedfrom human germline immunoglobulin sequences. In certain embodiments,however, such recombinant human antibodies are subjected to in vitromutagenesis (or, when an animal transgenic for human Ig sequences isused, in vivo somatic mutagenesis) and thus the amino acid sequences ofthe VH and VL regions of the recombinant antibodies are sequences that,while derived from and related to human germline VH and VL sequences,may not naturally exist within the human antibody germline repertoire invivo. One embodiment of the disclosure provides fully human antibodiescapable of binding human transferrin receptor which can be generatedusing techniques well known in the art, such as, but not limited to,using human Ig phage libraries such as those disclosed in Jermutus etal., PCT publication No. WO 2005/007699 A2.

Region of complementarity: As used herein, the term “region ofcomplementarity” refers to a nucleotide sequence, e.g., of anoligonucleotide, that is sufficiently complementary to a cognatenucleotide sequence, e.g., of a target nucleic acid, such that the twonucleotide sequences are capable of annealing to one another underphysiological conditions (e.g., in a cell). In some embodiments, aregion of complementarity is fully complementary to a cognate nucleotidesequence of target nucleic acid. However, in some embodiments, a regionof complementarity is partially complementary to a cognate nucleotidesequence of target nucleic acid (e.g., at least 80%, 90%, 95% or 99%complementarity). In some embodiments, a region of complementaritycontains 1, 2, 3, or 4 mismatches compared with a cognate nucleotidesequence of a target nucleic acid.

Specifically binds: As used herein, the term “specifically binds” refersto the ability of a molecule to bind to a binding partner with a degreeof affinity or avidity that enables the molecule to be used todistinguish the binding partner from an appropriate control in a bindingassay or other binding context. With respect to an antibody, the term,“specifically binds”, refers to the ability of the antibody to bind to aspecific antigen with a degree of affinity or avidity, compared with anappropriate reference antigen or antigens, that enables the antibody tobe used to distinguish the specific antigen from others, e.g., to anextent that permits preferential targeting to certain cells, e.g.,muscle cells, through binding to the antigen, as described herein. Insome embodiments, an antibody specifically binds to a target if theantibody has a K_(D) for binding the target of at least about 10⁻⁴ M,10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, 10⁻¹² M, 10⁻¹³M, or less. In some embodiments, an antibody specifically binds to thetransferrin receptor, e.g., an epitope of the apical domain oftransferrin receptor.

Subject: As used herein, the term “subject” refers to a mammal. In someembodiments, a subject is non-human primate, or rodent. In someembodiments, a subject is a human. In some embodiments, a subject is apatient, e.g., a human patient that has or is suspected of having adisease. In some embodiments, the subject is a human patient who has oris suspected of having a disease resulting from adisease-associated-repeat expansion, e.g., in a DMPK allele.

Transferrin receptor: As used herein, the term, “transferrin receptor”(also known as TFRC, CD71, p90, TFR, or TFR1) refers to an internalizingcell surface receptor that binds transferrin to facilitate iron uptakeby endocytosis. In some embodiments, a transferrin receptor may be ofhuman (NCBI Gene ID 7037), non-human primate (e.g., NCBI Gene ID 711568or NCBI Gene ID 102136007), or rodent (e.g., NCBI Gene ID 22042) origin.In addition, multiple human transcript variants have been characterizedthat encoded different isoforms of the receptor (e.g., as annotatedunder GenBank RefSeq Accession Numbers: NP_001121620.1, NP_003225.2,NP_001300894.1, and NP_001300895.1).

2′-modified nucleoside: As used herein, the terms “2′-modifiednucleoside” and “2′-modified ribonucleoside” are used interchangeablyand refer to a nucleoside having a sugar moiety modified at the 2′position. In some embodiments, the 2′-modified nucleoside is a 2′-4′bicyclic nucleoside, where the 2′ and 4′ positions of the sugar arebridged (e.g., via a methylene, an ethylene, or a (S)-constrained ethylbridge). In some embodiments, the 2′-modified nucleoside is anon-bicyclic 2′-modified nucleoside, e.g., where the 2′ position of thesugar moiety is substituted. Non-limiting examples of 2′-modifiednucleosides include: 2′-deoxy, 2′-fluoro (2′-F), 2′-O-methyl (2′-O-Me),2′-O-methoxyethyl (2′-MOE), 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE),2′-O—N-methylacetamido (2′-O-NMA), locked nucleic acid (LNA,methylene-bridged nucleic acid), ethylene-bridged nucleic acid (ENA),and (S)-constrained ethyl-bridged nucleic acid (cEt). In someembodiments, the 2′-modified nucleosides described herein arehigh-affinity modified nucleotides and oligonucleotides comprising the2′-modified nucleotides have increased affinity to a target sequences,relative to an unmodified oligonucleotide. Examples of structures of2′-modified nucleosides are provided below:

II. Complexes

Provided herein are complexes that comprise a targeting agent, e.g. anantibody, covalently linked to a molecular payload. In some embodiments,a complex comprises a muscle-targeting antibody covalently linked to anoligonucleotide. A complex may comprise an antibody that specificallybinds a single antigenic site or that binds to at least two antigenicsites that may exist on the same or different antigens.

A complex may be used to modulate the activity or function of at leastone gene, protein, and/or (e.g., and) nucleic acid. In some embodiments,the molecular payload present with a complex is responsible for themodulation of a gene, protein, and/or (e.g., and) nucleic acids. Amolecular payload may be a small molecule, protein, nucleic acid,oligonucleotide, or any molecular entity capable of modulating theactivity or function of a gene, protein, and/or (e.g., and) nucleic acidin a cell. In some embodiments, a molecular payload is anoligonucleotide that targets a disease-associated repeat in musclecells.

In some embodiments, a complex comprises a muscle-targeting agent, e.g.an anti-transferrin receptor antibody, covalently linked to a molecularpayload, e.g. an antisense oligonucleotide that targets adisease-associated repeat, e.g. DMPK allele.

A. Muscle-Targeting Agents

Some aspects of the disclosure provide muscle-targeting agents, e.g.,for delivering a molecular payload to a muscle cell. In someembodiments, such muscle-targeting agents are capable of binding to amuscle cell, e.g., via specifically binding to an antigen on the musclecell, and delivering an associated molecular payload to the muscle cell.In some embodiments, the molecular payload is bound (e.g., covalentlybound) to the muscle targeting agent and is internalized into the musclecell upon binding of the muscle targeting agent to an antigen on themuscle cell, e.g., via endocytosis. It should be appreciated thatvarious types of muscle-targeting agents may be used in accordance withthe disclosure. For example, the muscle-targeting agent may comprise, orconsist of, a nucleic acid (e.g., DNA or RNA), a peptide (e.g., anantibody), a lipid (e.g., a microvesicle), or a sugar moiety (e.g., apolysaccharide). Exemplary muscle-targeting agents are described infurther detail herein, however, it should be appreciated that theexemplary muscle-targeting agents provided herein are not meant to belimiting.

Some aspects of the disclosure provide muscle-targeting agents thatspecifically bind to an antigen on muscle, such as skeletal muscle,smooth muscle, or cardiac muscle. In some embodiments, any of themuscle-targeting agents provided herein bind to (e.g., specifically bindto) an antigen on a skeletal muscle cell, a smooth muscle cell, and/or(e.g., and) a cardiac muscle cell.

By interacting with muscle-specific cell surface recognition elements(e.g., cell membrane proteins), both tissue localization and selectiveuptake into muscle cells can be achieved. In some embodiments, moleculesthat are substrates for muscle uptake transporters are useful fordelivering a molecular payload into muscle tissue. Binding to musclesurface recognition elements followed by endocytosis can allow evenlarge molecules such as antibodies to enter muscle cells. As anotherexample molecular payloads conjugated to transferrin or anti-transferrinreceptor antibodies can be taken up by muscle cells via binding totransferrin receptor, which may then be endocytosed, e.g., viaclathrin-mediated endocytosis.

The use of muscle-targeting agents may be useful for concentrating amolecular payload (e.g., oligonucleotide) in muscle while reducingtoxicity associated with effects in other tissues. In some embodiments,the muscle-targeting agent concentrates a bound molecular payload inmuscle cells as compared to another cell type within a subject. In someembodiments, the muscle-targeting agent concentrates a bound molecularpayload in muscle cells (e.g., skeletal, smooth, or cardiac musclecells) in an amount that is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 30, 40, 50, 60, 70, 80, 90, or 100 times greater than an amount innon-muscle cells (e.g., liver, neuronal, blood, or fat cells). In someembodiments, a toxicity of the molecular payload in a subject is reducedby at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95% when it is delivered tothe subject when bound to the muscle-targeting agent.

In some embodiments, to achieve muscle selectivity, a muscle recognitionelement (e.g., a muscle cell antigen) may be required. As one example, amuscle-targeting agent may be a small molecule that is a substrate for amuscle-specific uptake transporter. As another example, amuscle-targeting agent may be an antibody that enters a muscle cell viatransporter-mediated endocytosis. As another example, a muscle targetingagent may be a ligand that binds to cell surface receptor on a musclecell. It should be appreciated that while transporter-based approachesprovide a direct path for cellular entry, receptor-based targeting mayinvolve stimulated endocytosis to reach the desired site of action.

i. Muscle-Targeting Antibodies

In some embodiments, the muscle-targeting agent is an antibody.Generally, the high specificity of antibodies for their target antigenprovides the potential for selectively targeting muscle cells (e.g.,skeletal, smooth, and/or (e.g., and) cardiac muscle cells). Thisspecificity may also limit off-target toxicity. Examples of antibodiesthat are capable of targeting a surface antigen of muscle cells havebeen reported and are within the scope of the disclosure. For example,antibodies that target the surface of muscle cells are described inArahata K., et al. “Immunostaining of skeletal and cardiac musclesurface membrane with antibody against Duchenne muscular dystrophypeptide” Nature 1988; 333: 861-3; Song K. S., et al. “Expression ofcaveolin-3 in skeletal, cardiac, and smooth muscle cells. Caveolin-3 isa component of the sarcolemma and co-fractionates with dystrophin anddystrophin-associated glycoproteins” J Biol Chem 1996; 271: 15160-5; andWeisbart R. H. et al., “Cell type specific targeted intracellulardelivery into muscle of a monoclonal antibody that binds myosin IIb” MolImmunol. 2003 March, 39(13):78309; the entire contents of each of whichare incorporated herein by reference.

a. Anti-Transferrin Receptor Antibodies

Some aspects of the disclosure are based on the recognition that agentsbinding to transferrin receptor, e.g., anti-transferrin-receptorantibodies, are capable of targeting muscle cell. Transferrin receptorsare internalizing cell surface receptors that transport transferrinacross the cellular membrane and participate in the regulation andhomeostasis of intracellular iron levels. Some aspects of the disclosureprovide transferrin receptor binding proteins, which are capable ofbinding to transferrin receptor. Accordingly, aspects of the disclosureprovide binding proteins (e.g., antibodies) that bind to transferrinreceptor. In some embodiments, binding proteins that bind to transferrinreceptor are internalized, along with any bound molecular payload, intoa muscle cell. As used herein, an antibody that binds to a transferrinreceptor may be referred to interchangeably as an, transferrin receptorantibody, an anti-transferrin receptor antibody, or an anti-TfRantibody. Antibodies that bind, e.g. specifically bind, to a transferrinreceptor may be internalized into the cell, e.g. throughreceptor-mediated endocytosis, upon binding to a transferrin receptor.

It should be appreciated that anti-transferrin receptor antibodies maybe produced, synthesized, and/or (e.g., and) derivatized using severalknown methodologies, e.g. library design using phage display. Exemplarymethodologies have been characterized in the art and are incorporated byreference (Díez, P. et al. “High-throughput phage-display screening inarray format”, Enzyme and microbial technology, 2015, 79, 34-41;Christoph M. H. and Stanley, J. R. “Antibody Phage Display: Techniqueand Applications” J Invest Dermatol. 2014, 134:2; Engleman, Edgar (Ed.)“Human Hybridomas and Monoclonal Antibodies.” 1985, Springer). In otherembodiments, an anti-transferrin receptor antibody has been previouslycharacterized or disclosed. Antibodies that specifically bind totransferrin receptor are known in the art (see, e.g. U.S. Pat. No.4,364,934, filed Dec. 4, 1979, “Monoclonal antibody to a human earlythymocyte antigen and methods for preparing same”; U.S. Pat. No.8,409,573, filed Jun. 14, 2006, “Anti-CD71 monoclonal antibodies anduses thereof for treating malignant tumor cells”; U.S. Pat. No.9,708,406, filed May 20, 2014, “Anti-transferrin receptor antibodies andmethods of use”; U.S. Pat. No. 9,611,323, filed Dec. 19, 2014, “Lowaffinity blood brain barrier receptor antibodies and uses therefor”; WO2015/098989, filed Dec. 24, 2014, “Novel anti-Transferrin receptorantibody that passes through blood-brain barrier”; Schneider C. et al.“Structural features of the cell surface receptor for transferrin thatis recognized by the monoclonal antibody OKT9.” J Biol Chem. 1982,257:14, 8516-8522; Lee et al. “Targeting Rat Anti-Mouse TransferrinReceptor Monoclonal Antibodies through Blood-Brain Barrier in Mouse”2000, J Pharmacol. Exp. Ther., 292: 1048-1052).

Provided herein, in some aspects, are new anti-TfR antibodies for use asthe muscle targeting agents (e.g., in muscle targeting complexes). Insome embodiments, the anti-TfR antibody described herein binds totransferrin receptor with high specificity and affinity. In someembodiments, the anti-TfR antibody described herein specifically bindsto any extracellular epitope of a transferrin receptor or an epitopethat becomes exposed to an antibody. In some embodiments, anti-TfRantibodies provided herein bind specifically to transferrin receptorfrom human, non-human primates, mouse, rat, etc. In some embodiments,anti-TfR antibodies provided herein bind to human transferrin receptor.In some embodiments, the anti-TfR antibody described herein binds to anamino acid segment of a human or non-human primate transferrin receptor,as provided in SEQ ID NOs: 105-108. In some embodiments, the anti-TfRantibody described herein binds to an amino acid segment correspondingto amino acids 90-96 of a human transferrin receptor as set forth in SEQID NO: 105, which is not in the apical domain of the transferrinreceptor.

An example human transferrin receptor amino acid sequence, correspondingto NCBI sequence NP_003225.2 (transferrin receptor protein 1 isoform 1,Homo sapiens) is as follows:

(SEQ ID NO: 105)MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAVDEEENADNNTKANVTKPKRCSGSICYGTIAVIVFFLIGFMIGYLGYCKGVEPKTECERLAGTESPVREEPGEDFPAARRLYWDDLKRKLSEKLDSTDFTGTIKLLNENSYVPREAGSQKDENLALYVENQFREFKLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCRMVTSESKNVKLTVSNVLKEIKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSGVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQNVKHPVTGQFLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELIERIPELNKVARAAAEVAGQFVIKLTHDVELNLDYERYNSQLLSFVRDLNQYRADIKEMGLSLQWLYSARGDFFRATSRLTTDFGNAEKTDRFVMKKLNDRVMRVEYHFLSPYVSPKESPFRHVFWGSGSHTLPALLENLKLRKQNNGAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF.

An example non-human primate transferrin receptor amino acid sequence,corresponding to NCBI sequence NP_001244232.1 (transferrin receptorprotein 1, Macaca mulatta) is as follows:

(SEQ ID NO: 106)MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLGVDEEENTDNNTKPNGTKPKRCGGNICYGTIAVIIFFLIGFMIGYLGYCKGVEPKTECERLAGTESPAREEPEEDFPAAPRLYWDDLKRKLSEKLDTTDFTSTIKLLNENLYVPREAGSQKDENLALYIENQFREFKLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGGLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLDSPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVKADLSFFGHAHLGTGDPYTPGFPSFNHTQFPPSQSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCKMVTSENKSVKLTVSNVLKETKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSSVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQDVKHPVTGRSLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELVERIPELNKVARAAAEVAGQFVIKLTHDTELNLDYERYNSQLLLFLRDLNQYRADVKEMGLSLQWLYSARGDFFRATSRLTTDFRNAEKRDKFVMKKLNDRVMRVEYYFLSPYVSPKESPFRHVFWGSGSHTLSALLESLKLRRQNNSAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF

An example non-human primate transferrin receptor amino acid sequence,corresponding to NCBI sequence XP_005545315.1 (transferrin receptorprotein 1, Macaca fascicularis) is as follows:

(SEQ ID NO: 107)MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLGVDEEENTDNNTKANGTKPKRCGGNICYGTIAVIIFFLIGFMIGYLGYCKGVEPKTECERLAGTESPAREEPEEDFPAAPRLYWDDLKRKLSEKLDTTDFTSTIKLLNENLYVPREAGSQKDENLALYIENQFREFKLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGGLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLDSPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVKADLSFFGHAHLGTGDPYTPGFPSFNHTQFPPSQSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCKMVTSENKSVKLTVSNVLKETKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSSVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQDVKHPVTGRSLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELVERIPELNKVARAAAEVAGQFVIKLTHDTELNLDYERYNSQLLLFLRDLNQYRADVKEMGLSLQWLYSARGDFFRATSRLTTDFRNAEKRDKFVMKKLNDRVMRVEYYFLSPYVSPKESPFRHVFWGSGSHTLSALLESLKLRRQNNSAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF.

An example mouse transferrin receptor amino acid sequence, correspondingto NCBI sequence NP_001344227.1 (transferrin receptor protein 1, Musmusculus) is as follows:

(SEQ ID NO: 108)MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAADEEENADNNMKASVRKPKRFNGRLCFAAIALVIFFLIGFMSGYLGYCKRVEQKEECVKLAETEETDKSETMETEDVPTSSRLYWADLKTLLSEKLNSIEFADTIKQLSQNTYTPREAGSQKDESLAYYIENQFHEFKFSKVWRDEHYVKIQVKSSIGQNMVTIVQSNGNLDPVESPEGYVAFSKPTEVSGKLVHANFGTKKDFEELSYSVNGSLVIVRAGEITFAEKVANAQSFNAIGVLIYMDKNKFPVVEADLALFGHAHLGTGDPYTPGFPSFNHTQFPPSQSSGLPNIPVQTISRAAAEKLFGKMEGSCPARWNIDSSCKLELSQNQNVKLIVKNVLKERRILNIFGVIKGYEEPDRYVVVGAQRDALGAGVAAKSSVGTGLLLKLAQVFSDMISKDGFRPSRSIIFASWTAGDFGAVGATEWLEGYLSSLHLKAFTYINLDKVVLGTSNFKVSASPLLYTLMGKIMQDVKHPVDGKSLYRDSNWISKVEKLSFDNAAYPFLAYSGIPAVSFCFCEDADYPYLGTRLDTYEALTQKVPQLNQMVRTAAEVAGQLIIKLTHDVELNLDYEMYNSKLLSFMKDLNQFKTDIRDMGLSLQWLYSARGDYFRATSRLTTDFHNAEKTNRFVMREINDRIMKVEYHFLSPYVSPRESPFRHIFWGSGSHTLSALVENLKLRQKNITAFNETLFRNQLALATWTIQGVANALSGDIWNIDNEF

In some embodiments, an anti-transferrin receptor antibody binds to anamino acid segment of the receptor as follows:FVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCRMVTSESKNVKLTVSNVLKE (SEQ ID NO: 109) and does not inhibit the bindinginteractions between transferrin receptors and transferrin and/or (e.g.,and) human hemochromatosis protein (also known as HFE). In someembodiments, the anti-transferrin receptor antibody described hereindoes not bind an epitope in SEQ ID NO: 109.

Appropriate methodologies may be used to obtain and/or (e.g., and)produce antibodies, antibody fragments, or antigen-binding agents, e.g.,through the use of recombinant DNA protocols. In some embodiments, anantibody may also be produced through the generation of hybridomas (see,e.g., Kohler, G and Milstein, C. “Continuous cultures of fused cellssecreting antibody of predefined specificity” Nature, 1975, 256:495-497). The antigen-of-interest may be used as the immunogen in anyform or entity, e.g., recombinant or a naturally occurring form orentity. Hybridomas are screened using standard methods, e.g. ELISAscreening, to find at least one hybridoma that produces an antibody thattargets a particular antigen. Antibodies may also be produced throughscreening of protein expression libraries that express antibodies, e.g.,phage display libraries. Phage display library design may also be used,in some embodiments, (see, e.g. U.S. Pat. No. 5,223,409, filed Mar. 1,1991, “Directed evolution of novel binding proteins”; WO 1992/18619,filed Apr. 10, 1992, “Heterodimeric receptor libraries using phagemids”;WO 1991/17271, filed May 1, 1991, “Recombinant library screeningmethods”; WO 1992/20791, filed May 15, 1992, “Methods for producingmembers of specific binding pairs”; WO 1992/15679, filed Feb. 28, 1992,and “Improved epitope displaying phage”). In some embodiments, anantigen-of-interest may be used to immunize a non-human animal, e.g., arodent or a goat. In some embodiments, an antibody is then obtained fromthe non-human animal, and may be optionally modified using a number ofmethodologies, e.g., using recombinant DNA techniques. Additionalexamples of antibody production and methodologies are known in the art(see, e.g. Harlow et al. “Antibodies: A Laboratory Manual”, Cold SpringHarbor Laboratory, 1988).

In some embodiments, an antibody is modified, e.g., modified viaglycosylation, phosphorylation, sumoylation, and/or (e.g., and)methylation. In some embodiments, an antibody is a glycosylatedantibody, which is conjugated to one or more sugar or carbohydratemolecules. In some embodiments, the one or more sugar or carbohydratemolecule are conjugated to the antibody via N-glycosylation,O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment),and/or (e.g., and) phosphoglycosylation. In some embodiments, the one ormore sugar or carbohydrate molecules are monosaccharides, disaccharides,oligosaccharides, or glycans. In some embodiments, the one or more sugaror carbohydrate molecule is a branched oligosaccharide or a branchedglycan. In some embodiments, the one or more sugar or carbohydratemolecule includes a mannose unit, a glucose unit, an N-acetylglucosamineunit, an N-acetylgalactosamine unit, a galactose unit, a fucose unit, ora phospholipid unit. In some embodiments, there are about 1-10, about1-5, about 5-10, about 1-4, about 1-3, or about 2 sugar molecules. Insome embodiments, a glycosylated antibody is fully or partiallyglycosylated. In some embodiments, an antibody is glycosylated bychemical reactions or by enzymatic means. In some embodiments, anantibody is glycosylated in vitro or inside a cell, which may optionallybe deficient in an enzyme in the N- or O-glycosylation pathway, e.g. aglycosyltransferase. In some embodiments, an antibody is functionalizedwith sugar or carbohydrate molecules as described in InternationalPatent Application Publication WO2014065661, published on May 1, 2014,entitled, “Modified antibody, antibody-conjugate and process for thepreparation thereof”.

In some embodiments, the anti-TfR antibody of the present disclosurecomprises a VL domain and/or (e.g., and) VH domain of any one of theanti-TfR antibodies selected from Table 2, and comprises a constantregion comprising the amino acid sequences of the constant regions of anIgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule, any class (e.g.,IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), or any subclass (e.g., IgG2a andIgG2b) of immunoglobulin molecule. Non-limiting examples of humanconstant regions are described in the art, e.g., see Kabat E A et al.,(1991) supra.

In some embodiments, agents binding to transferrin receptor, e.g.,anti-TfR antibodies, are capable of targeting muscle cell and/or (e.g.,and) mediate the transportation of an agent across the blood brainbarrier. Transferrin receptors are internalizing cell surface receptorsthat transport transferrin across the cellular membrane and participatein the regulation and homeostasis of intracellular iron levels. Someaspects of the disclosure provide transferrin receptor binding proteins,which are capable of binding to transferrin receptor. Antibodies thatbind, e.g. specifically bind, to a transferrin receptor may beinternalized into the cell, e.g. through receptor-mediated endocytosis,upon binding to a transferrin receptor.

Provided herein, in some aspects, are humanized antibodies that bind totransferrin receptor with high specificity and affinity. In someembodiments, the humanized anti-TfR antibody described hereinspecifically binds to any extracellular epitope of a transferrinreceptor or an epitope that becomes exposed to an antibody. In someembodiments, the humanized anti-TfR antibodies provided herein bindspecifically to transferrin receptor from human, non-human primates,mouse, rat, etc. In some embodiments, the humanized anti-TfR antibodiesprovided herein bind to human transferrin receptor. In some embodiments,the humanized anti-TfR antibody described herein binds to an amino acidsegment of a human or non-human primate transferrin receptor, asprovided in SEQ ID NOs: 105-108. In some embodiments, the humanizedanti-TfR antibody described herein binds to an amino acid segmentcorresponding to amino acids 90-96 of a human transferrin receptor asset forth in SEQ ID NO: 105, which is not in the apical domain of thetransferrin receptor. In some embodiments, the humanized anti-TfRantibodies described herein binds to TfR1 but does not bind to TfR2.

In some embodiments, an anti-TFR antibody specifically binds a TfR1(e.g., a human or non-human primate TfR1) with binding affinity (e.g.,as indicated by Kd) of at least about 10⁻⁴ M, 10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M,10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, 10⁻¹² M, 10⁻¹³ M, or less. In someembodiments, the anti-TfR antibodies described herein binds to TfR1 witha KD of sub-nanomolar range. In some embodiments, the anti-TfRantibodies described herein selectively binds to transferrin receptor 1(TfR1) but do not bind to transferrin receptor 2 (TfR2). In someembodiments, the anti-TfR antibodies described herein binds to humanTfR1 and cyno TfR1 (e.g., with a Kd of 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M,10⁻¹¹ M, 10⁻¹² M, 10⁻¹³ M, or less), but does not bind to a mouse TfR1.The affinity and binding kinetics of the anti-TfR antibody can be testedusing any suitable method including but not limited to biosensortechnology (e.g., OCTET or BIACORE). In some embodiments, binding of anyone of the anti-TfR antibody described herein does not complete with orinhibit transferrin binding to the TfR1. In some embodiments, binding ofany one of the anti-TfR antibody described herein does not complete withor inhibit HFE-beta-2-microglobulin binding to the TfR1.

The anti-TfR antibodies described herein are humanized antibodies. TheCDR and variable region amino acid sequences of the mouse monoclonalanti-TfR antibody from which the humanized anti-TfR antibodies describedherein are derived are provided in Table 2.

TABLE 2 Mouse Monoclonal Anti-TfR Antibodies No. System Ab IMGT KabatChothia 3-A4 CDR- GFNIKDDY (SEQ ID NO: DDYMY (SEQ ID NO: 7)GFNIKDD (SEQ ID H1 1) NO: 12) CDR- IDPENGDT (SEQ ID NO:WIDPENGDTEYASKFQD ENG (SEQ ID NO: H2 2) (SEQ ID NO: 8) 13) CDR-TLWLRRGLDY (SEQ ID WLRRGLDY (SEQ ID NO: 9) LRRGLD (SEQ ID H3 NO: 3)NO: 14) CDR- KSLLHSNGYTY (SEQ ID RSSKSLLHSNGYTYLF (SEQ SKSLLHSNGYTY L1NO: 4) ID NO: 10) (SEQ ID NO: 15) CDR- RMS (SEQ ID NO: 5)RMSNLAS (SEQ ID NO: 11) RMS (SEQ ID NO: L2 5) CDR- MQHLEYPFT (SEQ IDMQHLEYPFT (SEQ ID NO: 6) HLEYPF (SEQ ID L3 NO: 6) NO: 16) VHEVQLQQSGAELVRPGASVKLSCTASGFNIKDDYMYWVKQRPEQGLEWIGWIDPENGDTEYASKFQDKATVTADTSSNTAYLQLSSLTSEDTAVYYCTLWLRRGLDYWGQGTSVTVSS (SEQ ID NO: 17) VLDIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGYTYLFWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGGGTKLEIK (SEQ ID NO: 18) 3-A4 CDR- GFNIKDDY (SEQ ID NO: DDYMY (SEQ ID NO: 7)GFNIKDD (SEQ ID N54T* H1 1) NO: 12) CDR- IDPETGDT (SEQ ID NO:WIDPETGDTEYASKFQD ETG (SEQ ID NO: H2 19) (SEQ ID NO: 20) 21) CDR-TLWLRRGLDY (SEQ ID WLRRGLDY (SEQ ID NO: 9) LRRGLD (SEQ ID H3 NO: 3)NO: 14) CDR- KSLLHSNGYTY (SEQ ID RSSKSLLHSNGYTYLF (SEQ SKSLLHSNGYTY L1NO: 4) ID NO: 10) (SEQ ID NO: 15) CDR- RMS (SEQ ID NO: 5)RMSNLAS (SEQ ID NO: 11) RMS(SEQ ID NO: L2 5) CDR- MQHLEYPFT (SEQ IDMQHLEYPFT (SEQ ID NO: 6) HLEYPF (SEQ ID L3 NO: 6) NO: 16) VHEVQLQQSGAELVRPGASVKLSCTASGFNIKDDYMYWVKQRPEQGLEWIGWIDPETGDTEYASKFQDKATVTADTSSNTAYLQLSSLTSEDTAVYYCTLWLRRGLDYWGQGTSVTVSS (SEQ ID NO: 22) VLDIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGYTYLFWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGGGTKLEIK (SEQ ID NO: 18) 3-A4 CDR- GFNIKDDY (SEQ ID NO: DDYMY (SEQ ID NO: 7)GFNIKDD (SEQ ID N54S* H1 1) NO: 12) CDR- IDPESGDT (SEQ ID NO:WIDPESGDTEYASKFQD ESG (SEQ ID NO: H2 23) (SEQ ID NO: 24) 25) CDR-TLWLRRGLDY (SEQ ID WLRRGLDY (SEQ ID NO: 9) LRRGLD (SEQ ID H3 NO: 3)NO: 14) CDR- KSLLHSNGYTY (SEQ ID RSSKSLLHSNGYTYLF (SEQ SKSLLHSNGYTY L1NO: 4) ID NO: 10) (SEQ ID NO: 15) CDR- RMS (SEQ ID NO: 5)RMSNLAS (SEQ ID NO: 11) RMS (SEQ ID NO: L2 5) CDR- MQHLEYPFT (SEQ IDMQHLEYPFT (SEQ ID NO: 6) HLEYPF (SEQ ID L3 NO: 6) NO: 16) VHEVQLQQSGAELVRPGASVKLSCTASGFNIKDDYMYWVKQRPEQGLEWIGWIDPESGDTEYASKFQDKATVTADTSSNTAYLQLSSLTSEDTAVYYCTLWLRRGLDYWGQGTSVTVSS (SEQ ID NO: 26) VLDIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGYTYLFWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGGGTKLEIK (SEQ ID NO: 18) 3-M12 CDR- GYSITSGYY (SEQ ID SGYYWN (SEQ ID NO: 33)GYSITSGY (SEQ H1 NO: 27) ID NO: 38) CDR- ITFDGAN (SEQ ID NO:YITFDGANNYNPSLKN (SEQ FDG (SEQ ID NO: H2 28) ID NO: 34) 39) CDR-TRSSYDYDVLDY (SEQ SSYDYDVLDY (SEQ ID NO: SYDYDVLD (SEQ H3 ID NO: 29) 35)ID NO: 40) CDR- QDISNF (SEQ ID NO: 30) RASQDISNFLN (SEQ ID NO:SQDISNF (SEQ ID L1 36) NO: 41) CDR- YTS (SEQ ID NO: 31)YTSRLHS (SEQ ID NO: 37) YTS (SEQ ID NO: L2 31) CDR- QQGHTLPYT (SEQ IDQQGHTLPYT (SEQ ID NO: 32) GHTLPY (SEQ ID L3 NO: 32) NO: 42) VHDVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYITFDGANNYNPSLKNRISITRDTSKNQFFLKLTSVTTEDTATYYCTRSSYDYDVLDYWGQGTTLTVSS (SEQ ID NO: 43) VLDIQMTQTTSSLSASLGDRVTISCRASQDISNFLNWYQQRPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDFSLTVSNLEQEDIATYFCQQGHTLPYTFGGGTKLEIK (SEQ ID NO: 44)5-H12 CDR- GYSFTDYC (SEQ ID NO: DYCIN (SEQ ID NO: 51) GYSFTDY (SEQ ID H145) NO: 56) CDR- IYPGSGNT (SEQ ID NO: WIYPGSGNTRYSERFKG GSG (SEQ ID NO:H2 46) (SEQ ID NO: 52) 57) CDR- AREDYYPYHGMDY EDYYPYHGMDY (SEQ IDDYYPYHGMD H3 (SEQ ID NO: 47) NO: 53) (SEQ ID NO: 58) CDR-ESVDGYDNSF (SEQ ID RASESVDGYDNSFMH (SEQ SESVDGYDNSF Ll NO: 48)ID NO: 54) (SEQ ID NO: 59) CDR- RAS (SEQ ID NO: 49)RASNLES (SEQ ID NO: 55) RAS (SEQ ID NO: L2 49) CDR- QQSSEDPWT (SEQ IDQQSSEDPWT (SEQ ID NO: 50) SSEDPW (SEQ ID L3 NO: 50) NO: 60) VHQIQLQQSGPELVRPGASVKISCKASGYSFTDYCINWVNQRPGQGLEWIGWIYPGSGNTRYSERFKGKATLTVDTSSNTAYMQLSSLTSEDSAVYFCAREDYYPYHGMDYWGQGTSVTVSS (SEQ ID NO: 61) VLDIVLTQSPTSLAVSLGQRATISCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGIPARFSGSGSRTDFTLTINPVEAADVATYYCQQSSEDPWTFGGGTKLEIK (SEQ ID NO: 62) 5-H12 CDR- GYSFTDYY (SEQ ID DYYIN (SEQ ID NO: 64)GYSFTDY (SEQ ID C33Y* H1 NO: 63) NO: 56) CDR- IYPGSGNT (SEQ ID NO:WIYPGSGNTRYSERFKG GSG (SEQ ID NO: H2 46) (SEQ ID NO: 52) 57) CDR-AREDYYPYHGMDY EDYYPYHGMDY (SEQ ID DYYPYHGMD H3 (SEQ ID NO: 47) NO: 53)(SEQ ID NO: 58) CDR- ESVDGYDNSF (SEQ ID RASESVDGYDNSFMH (SEQ SESVDGYDNSFL1 NO: 48) ID NO: 54) (SEQ ID NO: 59) CDR- RAS (SEQ ID NO: 49)RASNLES (SEQ ID NO: 55) RAS (SEQ ID NO: L2 49) CDR- QQSSEDPWT (SEQ IDQQSSEDPWT (SEQ ID NO: 50) SSEDPW (SEQ ID L3 NO: 50) NO: 60) VHQIQLQQSGPELVRPGASVKISCKASGYSFTDYYINWVNQRPGQGLEWIGWIYPGSGNTRYSERFKGKATLTVDTSSNTAYMQLSSLTSEDSAVYFCAREDYYPYHGMDYWGQGTSVTVSS (SEQ ID NO: 65) VLDIVLTQSPTSLAVSLGQRATISCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGIPARFSGSGSRTDFTLTINPVEAADVATYYCQQSSEDPWTFGGGTKLEIK (SEQ ID NO: 62) 5-H12 CDR- GYSFTDYD (SEQ ID DYDIN (SEQ ID NO: 67)GYSFTDY (SEQ ID C33D* H1 NO: 66) NO: 56) CDR- IYPGSGNT (SEQ ID NO:WIYPGSGNTRYSERFKG GSG (SEQ ID NO: H2 46) (SEQ ID NO: 52) 57) CDR-AREDYYPYHGMDY EDYYPYHGMDY (SEQ ID DYYPYHGMD H3 (SEQ ID NO: 47) NO: 53)(SEQ ID NO: 58) CDR- ESVDGYDNSF (SEQ ID RASESVDGYDNSFMH (SEQ SESVDGYDNSFL1 NO: 48) ID NO: 54) (SEQ ID NO: 59) CDR- RAS (SEQ ID NO: 49)RASNLES (SEQ ID NO: 55) RAS (SEQ ID NO: L2 49) CDR- QQSSEDPWT (SEQ IDQQSSEDPWT (SEQ ID NO: 50) SSEDPW (SEQ ID L3 NO: 50) NO: 60) VHQIQLQQSGPELVRPGASVKISCKASGYSFTDYDINWVNQRPGQGLEWIGWIYPGS GNTRYSERFKGKATLTVDTSSNTAYMQLSSLTSEDSAVYFCAREDYYPYHGMDYWGQGTSVTVSS (SEQ ID NO: 68) VLDIVLTQSPTSLAVSLGQRATISCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGIPARFSGSGSRTDFTLTINPVEAADVATYYCQQSSEDPWTFGGGTKLEIK (SEQ ID NO: 62) *mutation positions are according to Kabat numberingof the respective VH sequences containing the mutations

In some embodiments, the anti-TfR antibody of the present disclosure isa humanized variant of any one of the anti-TfR antibodies provided inTable 2. In some embodiments, the anti-TfR antibody of the presentdisclosure comprises a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2,and a CDR-L3 that are the same as the CDR-H1, CDR-H2, and CDR-H3 in anyone of the anti-TfR antibodies provided in Table 2, and comprises ahumanized heavy chain variable region and/or (e.g., and) a humanizedlight chain variable region.

Humanized antibodies are human immunoglobulins (recipient antibody) inwhich residues from a complementarity determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat, or rabbit having the desiredspecificity, affinity, and capacity. In some embodiments, Fv frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, the humanized antibodymay comprise residues that are found neither in the recipient antibodynor in the imported CDR or framework sequences, but are included tofurther refine and optimize antibody performance. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin consensus sequence. The humanized antibody optimally alsowill comprise at least a portion of an immunoglobulin constant region ordomain (Fc), typically that of a human immunoglobulin. Antibodies mayhave Fc regions modified as described in WO 99/58572. Other forms ofhumanized antibodies have one or more CDRs (one, two, three, four, five,six) which are altered with respect to the original antibody, which arealso termed one or more CDRs derived from one or more CDRs from theoriginal antibody. Humanized antibodies may also involve affinitymaturation.

Humanized antibodies and methods of making them are known, e.g., asdescribed in Almagro et al., Front. Biosci. 13:1619-1633 (2008);Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'lAcad. Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337,7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34(2005); Padlan et al., Mol. Immunol. 28:489-498 (1991); Dall'Acqua etal., Methods 36:43-60 (2005); Osbourn et al., Methods 36:61-68 (2005);and Klimka et al., Br. J. Cancer, 83:252-260 (2000), the contents of allof which are incorporated herein by reference. Human framework regionsthat may be used for humanization are described in e.g., Sims et al. J.Immunol. 151:2296 (1993); Carter et al., Proc. Natl. Acad. Sci. USA,89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993); Almagro etal., Front. Biosci. 13:1619-1633 (2008)); Baca et al., J. Biol. Chem.272:10678-10684 (1997); and Rosok et al., J Biol. Chem. 271:22611-22618(1996), the contents of all of which are incorporated herein byreference.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising one or more amino acidvariations (e.g., in the VH framework region) as compared with any oneof the VHs listed in Table 2, and/or (e.g., and) a humanized VLcomprising one or more amino acid variations (e.g., in the VL frameworkregion) as compared with any one of the VLs listed in Table 2.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH containing no more than 25 aminoacid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) in the framework regions as compared with the VH of any ofthe anti-TfR antibodies listed in Table 2 (e.g., any one of SEQ ID NOs:17, 22, 26, 43, 61, 65, and 68). Alternatively or in addition (e.g., inaddition), the humanized anti-TfR antibody of the present disclosurecomprises a humanized VL containing no more than 25 amino acidvariations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) in the framework regions as compared with the VL of any oneof the anti-TfR antibodies listed in Table 2 (e.g., any one of SEQ IDNOs: 18, 44, and 62).

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising an amino acid sequencethat is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%)identical in the framework regions to the VH of any of the anti-TfRantibodies listed in Table 2 (e.g., any one of SEQ ID NOs: 17, 22, 26,43, 61, 65, and 68). Alternatively or in addition (e.g., in addition),In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VL comprising an amino acid sequencethat is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%)identical in the framework regions to the VL of any of the anti-TfRantibodies listed in Table 2 (e.g., any one of SEQ ID NOs: 18, 44, and62).

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 1 (according to the IMGT definition system),a CDR-H2 having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 19,or SEQ ID NO: 23 (according to the IMGT definition system), a CDR-H3having the amino acid sequence of SEQ ID NO: 3 (according to the IMGTdefinition system), and containing no more than 25 amino acid variations(e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in theframework regions as compared with the VH as set forth in SEQ ID NO: 17,SEQ ID NO: 22, or SEQ ID NO: 26. Alternatively or in addition (e.g., inaddition), the anti-TfR antibody of the present disclosure comprises ahumanized VL comprising a CDR-L1 having the amino acid sequence of SEQID NO: 4 (according to the IMGT definition system), a CDR-L2 having theamino acid sequence of SEQ ID NO: 5 (according to the IMGT definitionsystem), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 6(according to the IMGT definition system), and containing no more than25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19,18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 aminoacid variation) in the framework regions as compared with the VL as setforth in SEQ ID NO: 18.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 1 (according to the IMGT definition system),a CDR-H2 having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 19,or SEQ ID NO: 23 (according to the IMGT definition system), a CDR-H3having the amino acid sequence of SEQ ID NO: 3 (according to the IMGTdefinition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%,98%, or 99%) identical in the framework regions to the VH as set forthin SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 26. Alternatively or inaddition (e.g., in addition), the humanized anti-TfR antibody of thepresent disclosure comprises a humanized VL comprising a CDR-L1 havingthe amino acid sequence of SEQ ID NO: 4 (according to the IMGTdefinition system), a CDR-L2 having the amino acid sequence of SEQ IDNO: 5 (according to the IMGT definition system), and a CDR-L3 having theamino acid sequence of SEQ ID NO: 6 (according to the IMGT definitionsystem), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or99%) identical in the framework regions to the VL as set forth in anyone of SEQ ID NO: 18.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 7 (according to the Kabat definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 8, SEQ IDNO: 20, or SEQ ID NO: 24 (according to the Kabat definition system), aCDR-H3 having the amino acid sequence of SEQ ID NO: 9 (according to theKabat definition system), and containing no more than 25 amino acidvariations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) in the framework regions as compared with the VH as set forthin SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 26. Alternatively or inaddition (e.g., in addition), the humanized anti-TfR antibody of thepresent disclosure comprises a humanized VL comprising a CDR-L1 havingthe amino acid sequence of SEQ ID NO: 10 (according to the Kabatdefinition system), a CDR-L2 having the amino acid sequence of SEQ IDNO: 11 (according to the Kabat definition system), and a CDR-L3 havingthe amino acid sequence of SEQ ID NO: 6 (according to the Kabatdefinition system), and containing no more than 25 amino acid variations(e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in theframework regions as compared with the VL as set forth in SEQ ID NO: 18.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 7 (according to the Kabat definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 8, SEQ IDNO: 20, or SEQ ID NO: 24 (according to the Kabat definition system), aCDR-H3 having the amino acid sequence of SEQ ID NO: 9 (according to theKabat definition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%,95%, 98%, or 99%) identical in the framework regions to the VH as setforth in SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 26. Alternativelyor in addition (e.g., in addition), the humanized anti-TfR antibody ofthe present disclosure comprises a humanized VL comprising a CDR-L1having the amino acid sequence of SEQ ID NO: 10 (according to the Kabatdefinition system), a CDR-L2 having the amino acid sequence of SEQ IDNO: 11 (according to the Kabat definition system), and a CDR-L3 havingthe amino acid sequence of SEQ ID NO: 6 (according to the Kabatdefinition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%,98%, or 99%) identical in the framework regions to the VL as set forthin any one of SEQ ID NO: 18.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 12 (according to the Chothia definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 13, SEQID NO: 21, or SEQ ID NO: 25 (according to the Chothia definitionsystem), a CDR-H3 having the amino acid sequence of SEQ ID NO: 14(according to the Chothia definition system), and containing no morethan 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21,20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1amino acid variation) in the framework regions as compared with the VHas set forth in SEQ ID NO: 17, SEQ ID NO: 22 or SEQ ID NO: 26.Alternatively or in addition (e.g., in addition), the humanized anti-TfRantibody of the present disclosure comprises a humanized VL comprising aCDR-L1 having the amino acid sequence of SEQ ID NO: 15 (according to theChothia definition system), a CDR-L2 having the amino acid sequence ofSEQ ID NO: 5 (according to the Chothia definition system), and a CDR-L3having the amino acid sequence of SEQ ID NO: 16 (according to theChothia definition system), and containing no more than 25 amino acidvariations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) in the framework regions as compared with the VL as set forthin SEQ ID NO: 18.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 12 (according to the Chothia definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 13, SEQID NO: 21, or SEQ ID NO: 25 (according to the Chothia definitionsystem), a CDR-H3 having the amino acid sequence of SEQ ID NO: 14(according to the Chothia definition system), and is at least 75% (e.g.,75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regionsto the VH as set forth in SEQ ID NO: SEQ ID NO: 17, SEQ ID NO: 22 or SEQID NO: 26. Alternatively or in addition (e.g., in addition), theanti-TfR antibody of the present disclosure comprises a humanized VLcomprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 15(according to the Chothia definition system), a CDR-L2 having the aminoacid sequence of SEQ ID NO: 5 (according to the Chothia definitionsystem), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 16(according to the Chothia definition system), and is at least 75% (e.g.,75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regionsto the VL as set forth in any one of SEQ ID NO: 18.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 27 (according to the IMGT definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 28(according to the IMGT definition system), a CDR-H3 having the aminoacid sequence of SEQ ID NO: 29 (according to the IMGT definitionsystem), and containing no more than 25 amino acid variations (e.g., nomore than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the frameworkregions as compared with the VH as set forth in SEQ ID NO: 43.Alternatively or in addition (e.g., in addition), the humanized anti-TfRantibody of the present disclosure comprises a humanized VL comprising aCDR-L1 having the amino acid sequence of SEQ ID NO: 30 (according to theIMGT definition system), a CDR-L2 having the amino acid sequence of SEQID NO: 31 (according to the IMGT definition system), and a CDR-L3 havingthe amino acid sequence of SEQ ID NO: 32 (according to the IMGTdefinition system), and containing no more than 25 amino acid variations(e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in theframework regions as compared with the VL as set forth in SEQ ID NO: 44.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 27 (according to the IMGT definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 28(according to the IMGT definition system), a CDR-H3 having the aminoacid sequence of SEQ ID NO: 29 (according to the IMGT definitionsystem), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or99%) identical in the framework regions to the VH as set forth in SEQ IDNO: 43. Alternatively or in addition (e.g., in addition), the humanizedanti-TfR antibody of the present disclosure comprises a humanized VLcomprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 30(according to the IMGT definition system), a CDR-L2 having the aminoacid sequence of SEQ ID NO: 31 (according to the IMGT definitionsystem), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 32(according to the IMGT definition system), and is at least 75% (e.g.,75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regionsto the VL as set forth in SEQ ID NO: 44.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 33 (according to the Kabat definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 34(according to the Kabat definition system), a CDR-H3 having the aminoacid sequence of SEQ ID NO: 35 (according to the Kabat definitionsystem), and containing no more than 25 amino acid variations (e.g., nomore than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the frameworkregions as compared with the VH as set forth in SEQ ID NO: 43.Alternatively or in addition (e.g., in addition), the humanized anti-TfRantibody of the present disclosure comprises a humanized VL comprising aCDR-L1 having the amino acid sequence of SEQ ID NO: 36 (according to theKabat definition system), a CDR-L2 having the amino acid sequence of SEQID NO: 37 (according to the Kabat definition system), and a CDR-L3having the amino acid sequence of SEQ ID NO: 32 (according to the Kabatdefinition system), and containing no more than 25 amino acid variations(e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in theframework regions as compared with the VL as set forth in SEQ ID NO: 44.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 33 (according to the Kabat definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 34(according to the Kabat definition system), a CDR-H3 having the aminoacid sequence of SEQ ID NO: 35 (according to the Kabat definitionsystem), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or99%) identical in the framework regions to the VH as set forth in SEQ IDNO: 43. Alternatively or in addition (e.g., in addition), the humanizedanti-TfR antibody of the present disclosure comprises a humanized VLcomprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 36(according to the Kabat definition system), a CDR-L2 having the aminoacid sequence of SEQ ID NO: 37 (according to the Kabat definitionsystem), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 32(according to the Kabat definition system), and is at least 75% (e.g.,75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regionsto the VL as set forth in SEQ ID NO: 44.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 38 (according to the Chothia definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 39(according to the Chothia definition system), a CDR-H3 having the aminoacid sequence of SEQ ID NO: 40 (according to the Chothia definitionsystem), and containing no more than 25 amino acid variations (e.g., nomore than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the frameworkregions as compared with the VH as set forth in SEQ ID NO: 43.Alternatively or in addition (e.g., in addition), the humanized anti-TfRantibody of the present disclosure comprises a humanized VL comprising aCDR-L1 having the amino acid sequence of SEQ ID NO: 41 (according to theChothia definition system), a CDR-L2 having the amino acid sequence ofSEQ ID NO: 31 (according to the Chothia definition system), and a CDR-L3having the amino acid sequence of SEQ ID NO: 42 (according to theChothia definition system), and containing no more than 25 amino acidvariations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) in the framework regions as compared with the VL as set forthin SEQ ID NO: 44.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 38 (according to the Chothia definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 39(according to the Chothia definition system), a CDR-H3 having the aminoacid sequence of SEQ ID NO: 40 (according to the Chothia definitionsystem), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or99%) identical in the framework regions to the VH as set forth in SEQ IDNO: 43. Alternatively or in addition (e.g., in addition), the humanizedanti-TfR antibody of the present disclosure comprises a humanized VLcomprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 41(according to the Chothia definition system), a CDR-L2 having the aminoacid sequence of SEQ ID NO: 31 (according to the Chothia definitionsystem), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 42(according to the Chothia definition system), and is at least 75% (e.g.,75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regionsto the VL as set forth in SEQ ID NO: 44.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 45, SEQ ID NO: 63, or SEQ ID NO: 66(according to the IMGT definition system), a CDR-H2 having the aminoacid sequence of SEQ ID NO: 46 (according to the IMGT definitionsystem), a CDR-H3 having the amino acid sequence of SEQ ID NO: 47(according to the IMGT definition system), and containing no more than25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19,18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 aminoacid variation) in the framework regions as compared with the VH as setforth in SEQ ID NO: 61, SEQ ID NO: 65, or SEQ ID NO: 68. Alternativelyor in addition (e.g., in addition), the humanized anti-TfR antibody ofthe present disclosure comprises a humanized VL comprising a CDR-L1having the amino acid sequence of SEQ ID NO: 48 (according to the IMGTdefinition system), a CDR-L2 having the amino acid sequence of SEQ IDNO: 49 (according to the IMGT definition system), and a CDR-L3 havingthe amino acid sequence of SEQ ID NO: 50 (according to the IMGTdefinition system), and containing no more than 25 amino acid variations(e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in theframework regions as compared with the VL as set forth in SEQ ID NO: 62.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 45, SEQ ID NO: 63, or SEQ ID NO: 66(according to the IMGT definition system), a CDR-H2 having the aminoacid sequence of SEQ ID NO: 46 (according to the IMGT definitionsystem), a CDR-H3 having the amino acid sequence of SEQ ID NO: 47(according to the IMGT definition system), and is at least 75% (e.g.,75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regionsto the VH as set forth in SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 68.Alternatively or in addition (e.g., in addition), the humanized anti-TfRantibody of the present disclosure comprises a humanized VL comprising aCDR-L1 having the amino acid sequence of SEQ ID NO: 48 (according to theIMGT definition system), a CDR-L2 having the amino acid sequence of SEQID NO: 49 (according to the IMGT definition system), and a CDR-L3 havingthe amino acid sequence of SEQ ID NO: 50 (according to the IMGTdefinition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%,98%, or 99%) identical in the framework regions to the VL as set forthin SEQ ID NO: 62.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 51, SEQ ID NO: 64, or SEQ ID NO: 67(according to the Kabat definition system), a CDR-H2 having the aminoacid sequence of SEQ ID NO: 52 (according to the Kabat definitionsystem), a CDR-H3 having the amino acid sequence of SEQ ID NO: 53(according to the Kabat definition system), and containing no more than25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19,18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 aminoacid variation) in the framework regions as compared with the VH as setforth in SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 68. Alternatively orin addition (e.g., in addition), the humanized anti-TfR antibody of thepresent disclosure comprises a humanized VL comprising a CDR-L1 havingthe amino acid sequence of SEQ ID NO: 54 (according to the Kabatdefinition system), a CDR-L2 having the amino acid sequence of SEQ IDNO: 55 (according to the Kabat definition system), and a CDR-L3 havingthe amino acid sequence of SEQ ID NO: 50 (according to the Kabatdefinition system), and containing no more than 25 amino acid variations(e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in theframework regions as compared with the VL as set forth in SEQ ID NO: 62.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 51, SEQ ID NO: 64, or SEQ ID NO: 67(according to the Kabat definition system), a CDR-H2 having the aminoacid sequence of SEQ ID NO: 52 (according to the Kabat definitionsystem), a CDR-H3 having the amino acid sequence of SEQ ID NO: 53(according to the Kabat definition system), and is at least 75% (e.g.,75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regionsto the VH as set forth in SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 68.Alternatively or in addition (e.g., in addition), the humanized anti-TfRantibody of the present disclosure comprises a humanized VL comprising aCDR-L1 having the amino acid sequence of SEQ ID NO: 54 (according to theKabat definition system), a CDR-L2 having the amino acid sequence of SEQID NO: 55 (according to the Kabat definition system), and a CDR-L3having the amino acid sequence of SEQ ID NO: 50 (according to the Kabatdefinition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%,98%, or 99%) identical in the framework regions to the VL as set forthin SEQ ID NO: 62.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 56 (according to the Chothia definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 57(according to the Chothia definition system), a CDR-H3 having the aminoacid sequence of SEQ ID NO: 58 (according to the Chothia definitionsystem), and containing no more than 25 amino acid variations (e.g., nomore than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the frameworkregions as compared with the VH as set forth in SEQ ID NO: 61, SEQ IDNO: 65, SEQ ID NO: 68. Alternatively or in addition (e.g., in addition),the humanized anti-TfR antibody of the present disclosure comprises ahumanized VL comprising a CDR-L1 having the amino acid sequence of SEQID NO: 59 (according to the Chothia definition system), a CDR-L2 havingthe amino acid sequence of SEQ ID NO: 49 (according to the Chothiadefinition system), and a CDR-L3 having the amino acid sequence of SEQID NO: 60 (according to the Chothia definition system), and containingno more than 25 amino acid variations (e.g., no more than 25, 24, 23,22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,2, or 1 amino acid variation) in the framework regions as compared withthe VL as set forth in SEQ ID NO: 62.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 56 (according to the Chothia definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 57(according to the Chothia definition system), a CDR-H3 having the aminoacid sequence of SEQ ID NO: 58 (according to the Chothia definitionsystem), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or99%) identical in the framework regions to the VH as set forth in SEQ IDNO: 61, SEQ ID NO: 65, SEQ ID NO: 68. Alternatively or in addition(e.g., in addition), the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VL comprising a CDR-L1 having the aminoacid sequence of SEQ ID NO: 59 (according to the Chothia definitionsystem), a CDR-L2 having the amino acid sequence of SEQ ID NO: 49(according to the Chothia definition system), and a CDR-L3 having theamino acid sequence of SEQ ID NO: 60 (according to the Chothiadefinition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%,98%, or 99%) identical in the framework regions to the VL as set forthin SEQ ID NO: 62.

Examples of amino acid sequences of the humanized anti-TfR antibodiesdescribed herein are provided in Table 3.

TABLE 3 Variable Regions of Humanized Anti-TfR Antibodies AntibodyVariable Region Amino Acid Sequence** 3A4 V_(H): VH3 (N54T*)/Vκ4EVQLVQSGSELKKPGASVKVSCTASGFNIKDDYMYWVRQPPGKGLEWIGWIDPETGDTEYASKFQDRVTVTADTSTNTAYMELSSLRSEDTAVYYCTLWLRRGLDYWGQGTLVTVSS (SEQ ID NO: 69) V_(L):DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGYTYLFWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKVEIK (SEQ ID NO: 70) 3A4 V_(H): VH3 (N54S*)/Vκ4EVQLVQSGSELKKPGASVKVSCTASGFNIKDDYMYWVRQPPGKGLEWIGWIDPESGDTEYASKFQDRVTVTADTSTNTAYMELSSLRSEDTAVYYCTLWLRRGLDYWGQGTLVTVSS (SEQ ID NO: 71) V_(L):DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGYTYLFWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKVEIK (SEQ ID NO: 70) 3A4 V_(H): VH3/Vκ4EVQLVQSGSELKKPGASVKVSCTASGFNIKDDYMYWVRQPPGKGLEWIGWIDPENGDTEYASKFQDRVTVTADTSTNTAYMELSSLRSEDTAVYYCTLWLRRGLDYWGQGTLVTVSS (SEQ ID NO: 72) V_(L):DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGYTYLFWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKVEIK (SEQ ID NO: 70) 3M12 V_(H): VH3/Vκ2QVQLQESGPGLVKPSQTLSLTCSVTGYSITSGYYWNWIRQPPGKGLEWMGYITFDGANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSS (SEQ ID NO: 73) V_(L):DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGHTLPYTFGQGTKLEIK (SEQ ID NO: 74)3M12 V_(H): VH3/Vκ3QVQLQESGPGLVKPSQTLSLTCSVTGYSITSGYYWNWIRQPPGKGLEWMGYITFDGANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSS (SEQ ID NO: 73) V_(L):DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHTLPYTFGQGTKLEIK (SEQ ID NO: 75)3M12 V_(H): VH4/Vκ2QVQLQESGPGLVKPSQTLSLTCTVTGYSITSGYYWNWIRQPPGKGLEWIGYITFDGANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSS (SEQ ID NO: 76) V_(L):DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGHTLPYTFGQGTKLEIK (SEQ ID NO: 74)3M12 V_(H): VH4/Vκ3QVQLQESGPGLVKPSQTLSLTCTVTGYSITSGYYWNWIRQPPGKGLEWIGYITFDGANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSS (SEQ ID NO: 76) V_(L):DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHTLPYTFGQGTKLEIK (SEQ ID NO: 75)5H12 V_(H): VH5 (C33Y*)/Vκ3QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYYINWVRQAPGQGLEWMGWIYPGSGNTRYSERFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREDYYPYHGMDYWGQGTLVTVSS (SEQ ID NO: 77) V_(L):DIVLTQSPDSLAVSLGERATINCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGVPDRFSGSGSRTDFTLTISSLQAEDVAVYYCQQSSEDPWTFGQGTKLEIK (SEQ ID NO: 78) 5H12 V_(H): VH5 (C33D*)/Vκ4QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYDINWVRQAPGQGLEWMGWIYPGSGNTRYSERFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREDYYPYHGMDYWGQGTLVTVSS (SEQ ID NO: 79) V_(L):DIVMTQSPDSLAVSLGERATINCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSSEDPWTFGQGTKLEIK (SEQ ID NO: 80) 5H12 V_(H): VH5 (C33Y*)/Vκ4QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYYINWVRQAPGQGLEWMGWIYPGSGNTRYSERFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREDYYPYHGMDYWGQGTLVTVSS (SEQ ID NO: 77) V_(L):DIVMTQSPDSLAVSLGERATINCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSSEDPWTFGQGTKLEIK (SEQ ID NO: 80) *mutation positions are according to Kabat numberingof the respective VH sequences containing the mutations **CDRs accordingto the Kabat numbering system are bolded

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising the CDR-H1, CDR-H2, andCDR-H3 of any one of the anti-TfR antibodies provided in Table 2 andcomprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)amino acid variations in the framework regions as compared with therespective humanized VH provided in Table 3. Alternatively or inaddition (e.g., in addition), the humanized anti-TfR antibody of thepresent disclosure comprises a humanized VL comprising the CDR-L1,CDR-L2, and CDR-L3 of any one of the anti-TfR antibodies provided inTable 2 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more) amino acid variations in the framework regions as compared withthe respective humanized VL provided in Table 3.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 69, and/or (e.g., and) a humanized VL comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 70. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a humanized VHcomprising the amino acid sequence of SEQ ID NO: 69 and a humanized VLcomprising the amino acid sequence of SEQ ID NO: 70.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 71, and/or (e.g., and) a humanized VL comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 70. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a humanized VHcomprising the amino acid sequence of SEQ ID NO: 71 and a humanized VLcomprising the amino acid sequence of SEQ ID NO: 70.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 72, and/or (e.g., and) a humanized VL comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 70. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a humanized VHcomprising the amino acid sequence of SEQ ID NO: 72 and a humanized VLcomprising the amino acid sequence of SEQ ID NO: 70.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 73, and/or (e.g., and) a humanized VL comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 74. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a humanized VHcomprising the amino acid sequence of SEQ ID NO: 73 and a humanized VLcomprising the amino acid sequence of SEQ ID NO: 74.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 73, and/or (e.g., and) a humanized VL comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 75. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a humanized VHcomprising the amino acid sequence of SEQ ID NO: 73 and a humanized VLcomprising the amino acid sequence of SEQ ID NO: 75.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 76, and/or (e.g., and) a humanized VL comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 74. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a humanized VHcomprising the amino acid sequence of SEQ ID NO: 76 and a humanized VLcomprising the amino acid sequence of SEQ ID NO: 74.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 76, and/or (e.g., and) a humanized VL comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 75. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a humanized VHcomprising the amino acid sequence of SEQ ID NO: 76 and a humanized VLcomprising the amino acid sequence of SEQ ID NO: 75.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 77, and/or (e.g., and) a humanized VL comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 78. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a humanized VHcomprising the amino acid sequence of SEQ ID NO: 77 and a humanized VLcomprising the amino acid sequence of SEQ ID NO: 78.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 79, and/or (e.g., and) a humanized VL comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 80. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a humanized VHcomprising the amino acid sequence of SEQ ID NO: 79 and a humanized VLcomprising the amino acid sequence of SEQ ID NO: 80.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 77, and/or (e.g., and) a humanized VL comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 80. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a humanized VHcomprising the amino acid sequence of SEQ ID NO: 77 and a humanized VLcomprising the amino acid sequence of SEQ ID NO: 80.

In some embodiments, the humanized anti-TfR antibody described herein isa full-length IgG, which can include a heavy constant region and a lightconstant region from a human antibody. In some embodiments, the heavychain of any of the anti-TfR antibodies as described herein maycomprises a heavy chain constant region (CH) or a portion thereof (e.g.,CH1, CH2, CH3, or a combination thereof). The heavy chain constantregion can of any suitable origin, e.g., human, mouse, rat, or rabbit.In one specific example, the heavy chain constant region is from a humanIgG (a gamma heavy chain), e.g., IgG1, IgG2, or IgG4. An example of ahuman IgG1 constant region is given below:

(SEQ ID NO: 81) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG  K

In some embodiments, the heavy chain of any of the anti-TfR antibodiesdescribed herein comprises a mutant human IgG1 constant region. Forexample, the introduction of LALA mutations (a mutant derived from mAbb12 that has been mutated to replace the lower hinge residues Leu234Leu235 with Ala234 and Ala235) in the CH2 domain of human IgG1 is knownto reduce Fey receptor binding (Bruhns, P., et al. (2009) and Xu, D. etal. (2000)). The mutant human IgG1 constant region is provided below(mutations bonded and underlined):

(SEQ ID NO: 82) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPEAA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K

In some embodiments, the light chain of any of the anti-TfR antibodiesdescribed herein may further comprise a light chain constant region(CL), which can be any CL known in the art. In some examples, the CL isa kappa light chain. In other examples, the CL is a lambda light chain.In some embodiments, the CL is a kappa light chain, the sequence ofwhich is provided below:

(SEQ ID NO: 83) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL  SSPVTKSFNRGEC

Other antibody heavy and light chain constant regions are well known inthe art, e.g., those provided in the IMGT database (www.imgt.org) or atwww.vbase2.org/vbstat.php., both of which are incorporated by referenceherein.

In some embodiments, the humanized anti-TfR antibody described hereincomprises a heavy chain comprising any one of the VH as listed in Table3 or any variants thereof and a heavy chain constant region that is atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99%identical to SEQ ID NO: 81 or SEQ ID NO: 82. In some embodiments, thehumanized anti-TfR antibody described herein comprises a heavy chaincomprising any one of the VH as listed in Table 3 or any variantsthereof and a heavy chain constant region that contains no more than 25amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19,18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 aminoacid variation) as compared with SEQ ID NO: 81 or SEQ ID NO: 82. In someembodiments, the humanized anti-TfR antibody described herein comprisesa heavy chain comprising any one of the VH as listed in Table 3 or anyvariants thereof and a heavy chain constant region as set forth in SEQID NO: 81. In some embodiments, the humanized anti-TfR antibodydescribed herein comprises heavy chain comprising any one of the VH aslisted in Table 3 or any variants thereof and a heavy chain constantregion as set forth in SEQ ID NO: 82.

In some embodiments, the humanized anti-TfR antibody described hereincomprises a light chain comprising any one of the VL as listed in Table3 or any variants thereof and a light chain constant region that is atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99%identical to SEQ ID NO: 83. In some embodiments, the humanized anti-TfRantibody described herein comprises a light chain comprising any one ofthe VL as listed in Table 3 or any variants thereof and a light chainconstant region contains no more than 25 amino acid variations (e.g., nomore than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared withSEQ ID NO: 83. In some embodiments, the humanized anti-TfR antibodydescribed herein comprises a light chain comprising any one of the VL aslisted in Table 3 or any variants thereof and a light chain constantregion set forth in SEQ ID NO: 83.

Examples of IgG heavy chain and light chain amino acid sequences of theanti-TfR antibodies described are provided in Table 4 below.

TABLE 4Heavy chain and light chain sequences of examples of humanized anti-TfR IgGsAntibody IgG Heavy Chain/Light Chain Sequences** 3A4Heavy Chain (with wild type human IgG1 constant region) VH3 (N54T*)/Vκ4EVQLVQSGSELKKPGASVKVSCTASGFNIKDDYMYWVRqPPGKGLEWIGWIDPETGDTEYASKFQDRVTVTADTSTNTAYMELSSLRSEDTAVYYCTLWLRRGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 84)Light Chain (with kappa light chain constant region)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGYTYLFWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 85) 3A4Heavy Chain (with wild type human IgG1 constant region) VH3 (N54S*)/Vκ4EVQLVQSGSELKKPGASVKVSCTASGFNIKDDYMYWVRQPPGKGLEWIGWIDPESGDTEYASKFQDRVTVTADTSTNTAYMELSSLRSEDTAVYYCTLWLRRGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 86)Light Chain (with kappa light chain constant region)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGYTYLFWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 85) 3A4Heavy Chain (with wild type human IgG1 constant region) VH3/Vκ4EVQLVQSGSELKKPGASVKVSCTASGFNIKDDYMYWVRQPPGKGLEWIGWIDPENGDTEYASKFQDRVTVTADTSTNTAYMELSSLRSEDTAVYYCTLWLRRGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 87)Light Chain (with kappa light chain constant region)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGYTYLFWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 85)3M12 Heavy Chain (with wild type human IgG1 constant region) VH3/Vκ2QVQLQESGPGLVKPSQTLSLTCSVTGYSITSGYYWNWIRQPPGKGLEWMGYITFDGANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 88)Light Chain (with kappa light chain constant region)DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGHTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 89) 3M12Heavy Chain (with wild type human IgG1 constant region) VH3/Vκ3QVQLQESGPGLVKPSQTLSLTCSVTGYSITSGYYWNWIRQPPGKGLEWMGYITFDGANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 88)Light Chain (with kappa light chain constant region)DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 90) 3M12Heavy Chain (with wild type human IgG1 constant region) VH4/Vκ2QVQLQESGPGLVKPSQTLSLTCTVTGYSITSGYYWNWIRQPPGKGLEWIGYITFDGANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 91)Light Chain (with kappa light chain constant region)DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGHTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 89) 3M12Heavy Chain (with wild type human IgG1 constant region) VH4/Vκ3QVQLQESGPGLVKPSQTLSLTCTVTGYSITSGYYWNWIRQPPGKGLEWIGYITFDGANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 91)Light Chain (with kappa light chain constant region)DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 90) 5H12Heavy Chain (with wild type human IgG1 constant region) VH5 (C33Y*)/Vκ3QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYYINWVRQAPGQGLEWMGWIYPGSGNTRYSERFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREDYYPYHGMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 92)Light Chain (with kappa light chain constant region)DIVLTQSPDSLAVSLGERATINCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGVPDRFSGSGSRTDFTLTISSLQAEDVAVYYCQQSSEDPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 93) 5H12Heavy Chain (with wild type human IgG1 constant region) VH5 (C33D*)/Vκ4QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYDINWVRQAPGQGLEWMGWIYPGSGNTRYSERFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREDYYPYHGMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 94)Light Chain (with kappa light chain constant region)DIVMTQSPDSLAVSLGERATINCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSSEDPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 95)5H12 Heavy Chain (with wild type human IgG1 constant region)VH5 (C33Y*)/Vκ4 QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYYINWVRQAPGQGLEWMGWIYPGSGNTRYSERFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREDYYPYHGMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 92)Light Chain (with kappa light chain constant region)DIVMTQSPDSLAVSLGERATINCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSSEDPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 95)*mutation positions are according to Kabat numbering of the respectiveVH sequences containing the mutations **CDRs according to the Kabatnumbering system are bolded; VH/VL sequences underlined

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain containing no more than 25 amino acidvariations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) as compared with the heavy chain as set forth in any one ofSEQ ID NOs: 84, 86, 87, 88, 91, 92, and 94. Alternatively or in addition(e.g., in addition), the humanized anti-TfR antibody of the presentdisclosure comprises a light chain containing no more than 25 amino acidvariations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) as compared with the light chain as set forth in any one ofSEQ ID NOs: 85, 89, 90, 93, and 95.

In some embodiments, the humanized anti-TfR antibody described hereincomprises a heavy chain comprising an amino acid sequence that is atleast 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to anyone of SEQ ID NOs: 84, 86, 87, 88, 91, 92, and 94. Alternatively or inaddition (e.g., in addition), the humanized anti-TfR antibody describedherein comprises a light chain comprising an amino acid sequence that isat least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical toany one of SEQ ID NOs: 85, 89, 90, 93, and 95. In some embodiments, theanti-TfR antibody described herein comprises a heavy chain comprisingthe amino acid sequence of any one of SEQ ID NOs: 84, 86, 87, 88, 91,92, and 94. Alternatively or in addition (e.g., in addition), theanti-TfR antibody described herein comprises a light chain comprisingthe amino acid sequence of any one of SEQ ID NOs: 85, 89, 90, 93, and95.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 84, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 85. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 84 and a light chaincomprising the amino acid sequence of SEQ ID NO: 85.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 86, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 85. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 86 and a light chaincomprising the amino acid sequence of SEQ ID NO: 85.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 87, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 85. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 87 and a light chaincomprising the amino acid sequence of SEQ ID NO: 85.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 88, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 89. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 88 and a light chaincomprising the amino acid sequence of SEQ ID NO: 89.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 88, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 90. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 88 and a light chaincomprising the amino acid sequence of SEQ ID NO: 90.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 91, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 89. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 91 and a light chaincomprising the amino acid sequence of SEQ ID NO: 89.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 91, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 90. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 91 and a light chaincomprising the amino acid sequence of SEQ ID NO: 90.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 92, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 93. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 92 and a light chaincomprising the amino acid sequence of SEQ ID NO: 93.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 94, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 95. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 94 and a light chaincomprising the amino acid sequence of SEQ ID NO: 95.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 92, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 95. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 92 and a light chaincomprising the amino acid sequence of SEQ ID NO: 95.

In some embodiments, the anti-TfR antibody is a Fab fragment, Fab′fragment, or F(ab′)2 fragment of an intact antibody (full-lengthantibody). Antigen binding fragment of an intact antibody (full-lengthantibody) can be prepared via routine methods (e.g., recombinantly or bydigesting the heavy chain constant region of a full length IgG using anenzyme such as papain). For example, F(ab′)2 fragments can be producedby pepsin or papain digestion of an antibody molecule, and Fab′fragments that can be generated by reducing the disulfide bridges ofF(ab′)2 fragments. In some embodiments, a heavy chain constant region ina Fab fragment of the anti-TfR1 antibody described herein comprises theamino acid sequence of:

(SEQ ID NO: 96) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHT 

In some embodiments, the humanized anti-TfR antibody described hereincomprises a heavy chain comprising any one of the VH as listed in Table3 or any variants thereof and a heavy chain constant region that is atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99%identical to SEQ ID NO: 96. In some embodiments, the humanized anti-TfRantibody described herein comprises a heavy chain comprising any one ofthe VH as listed in Table 3 or any variants thereof and a heavy chainconstant region that contains no more than 25 amino acid variations(e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) ascompared with SEQ ID NO: 96. In some embodiments, the humanized anti-TfRantibody described herein comprises a heavy chain comprising any one ofthe VH as listed in Table 3 or any variants thereof and a heavy chainconstant region as set forth in SEQ ID NO: 96.

In some embodiments, the humanized anti-TfR antibody described hereincomprises a light chain comprising any one of the VL as listed in Table3 or any variants thereof and a light chain constant region that is atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99%identical to SEQ ID NO: 83. In some embodiments, the humanized anti-TfRantibody described herein comprises a light chain comprising any one ofthe VL as listed in Table 3 or any variants thereof and a light chainconstant region contains no more than 25 amino acid variations (e.g., nomore than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared withSEQ ID NO: 83. In some embodiments, the humanized anti-TfR antibodydescribed herein comprises a light chain comprising any one of the VL aslisted in Table 3 or any variants thereof and a light chain constantregion set forth in SEQ ID NO: 83.

Examples of Fab heavy chain and light chain amino acid sequences of theanti-TfR antibodies described are provided in Table 5 below.

TABLE 5Heavy chain and light chain sequences of examples of humanized anti-TfR FabsAntibody Fab Heavy Chain/Light Chain Sequences** 3A4Heavy Chain (with partial human IgG1 constant region) VH3 (N54T*)/Vκ4EVQLVQSGSELKKPGASVKVSCTASGFNIKDDYMYWVRQPPGKGLEWIGWIDPETGDTEYASKFQDRVTVTADTSTNTAYMELSSLRSEDTAVYYCTLWLRRGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 97)Light Chain (with kappa light chain constant region)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGYTYLFWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 85) 3A4Heavy Chain (with partial human IgG1 constant region) VH3 (N54S*)/Vκ4EVQLVQSGSELKKPGASVKVSCTASGFNIKDDYMYWVRQPPGKGLEWIGWIDPESGDTEYASKFQDRVTVTADTSTNTAYMELSSLRSEDTAVYYCTLWLRRGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 98)Light Chain (with kappa light chain constant region)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGYTYLFWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 85) 3A4Heavy Chain (with partial human IgG1 constant region) VH3/Vκ4EVQLVQSGSELKKPGASVKVSCTASGFNIKDDYMYWVRQPPGKGLEWIGWIDPENGDTEYASKFQDRVTVTADTSTNTAYMELSSLRSEDTAVYYCTLWLRRGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 99)Light Chain (with kappa light chain constant region)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGYTYLFWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 85) 3M12Heavy Chain (with partial human IgG1 constant region) VH3/Vκ2QVQLQESGPGLVKPSQTLSLTCSVTGYSITSGYYWNWIRQPPGKGLEWMGYITFDGANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 100)Light Chain (with kappa light chain constant region)DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGHTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 89)3M12 Heavy Chain (with partial human IgG1 constant region) VH3/Vκ3QVQLQESGPGLVKPSQTLSLTCSVTGYSITSGYYWNWIRQPPGKGLEWMGYITFDGANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 100)Light Chain (with kappa light chain constant region)DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 90)3M12 Heavy Chain (with partial human IgG1 constant region) VH4/Vκ2QVQLQESGPGLVKPSQTLSLTCTVTGYSITSGYYWNWIRQPPGKGLEWIGYITFDGANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 101)Light Chain (with kappa light chain constant region)DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGHTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 89)3M12 Heavy Chain (with partial human IgG1 constant region) VH4/Vκ3QVQLQESGPGLVKPSQTLSLTCTVTGYSITSGYYWNWIRQPPGKGLEWIGYITFDGANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 101)Light Chain (with kappa light chain constant region)DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 90)5H12 Heavy Chain (with partial human IgG1 constant region)VH5 (C33Y*)/Vκ3 QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYYINWVRQAPGQGLEWMGWIYPGSGNTRYSERFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREDYYPYHGMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 102)Light Chain (with kappa light chain constant region)DIVLTQSPDSLAVSLGERATINCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGVPDRFSGSGSRTDFTLTISSLQAEDVAVYYCQQSSEDPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 93) 5H12Heavy Chain (with partial human IgG1 constant region) VH5 (C33D*)/Vκ4QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYDINWVRQAPGQGLEWMGWIYPGSGNTRYSERFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREDYYPYHGMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NQ: 103)Light Chain (with kappa light chain constant region)DIVMTQSPDSLAVSLGERATINCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSSEDPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 95) 5H12Heavy Chain (with partial human IgG1 constant region) VH5 (C33Y*)/Vκ4QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYYINWVRQAPGQGLEWMGWIYPGSGNTRYSERFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREDYYPYHGMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 102)Light Chain (with kappa light chain constant region)DIVMTQSPDSLAVSLGERATINCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSSEDPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 95) *mutation positions are according to Kabatnumbering of the respective VH sequences containing the mutations **CDRsaccording to the Kabat numbering system are bolded; VH/VL sequencesunderlined

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain containing no more than 25 amino acidvariations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) as compared with the heavy chain as set forth in any one ofSEQ ID NOs: 97-103. Alternatively or in addition (e.g., in addition),the humanized anti-TfR antibody of the present disclosure comprises alight chain containing no more than 25 amino acid variations (e.g., nomore than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared withthe light chain as set forth in any one of SEQ ID NOs: 85, 89, 90, 93,and 95.

In some embodiments, the humanized anti-TfR antibody described hereincomprises a heavy chain comprising an amino acid sequence that is atleast 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to anyone of SEQ ID NOs: 97-103. Alternatively or in addition (e.g., inaddition), the humanized anti-TfR antibody described herein comprises alight chain comprising an amino acid sequence that is at least 75%(e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQID NOs: 85, 89, 90, 93, and 95. In some embodiments, the anti-TfRantibody described herein comprises a heavy chain comprising the aminoacid sequence of any one of SEQ ID NOs: 97-103. Alternatively or inaddition (e.g., in addition), the anti-TfR antibody described hereincomprises a light chain comprising the amino acid sequence of any one ofSEQ ID NOs: 85, 89, 90, 93, and 95.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 97, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 85. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 97 and a light chaincomprising the amino acid sequence of SEQ ID NO: 85.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 98, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 85. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 98 and a light chaincomprising the amino acid sequence of SEQ ID NO: 85.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 99, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 85. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 99 and a light chaincomprising the amino acid sequence of SEQ ID NO: 85.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 100, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 89. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 100 and a light chaincomprising the amino acid sequence of SEQ ID NO: 89.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 100, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 90. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 100 and a light chaincomprising the amino acid sequence of SEQ ID NO: 90.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 101, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 89. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 101 and a light chaincomprising the amino acid sequence of SEQ ID NO: 89.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 101, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 90. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 101 and a light chaincomprising the amino acid sequence of SEQ ID NO: 90.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 102, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 93. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 102 and a light chaincomprising the amino acid sequence of SEQ ID NO: 93.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 103, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 95. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 103 and a light chaincomprising the amino acid sequence of SEQ ID NO: 95.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 102, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 95. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 102 and a light chaincomprising the amino acid sequence of SEQ ID NO: 95.

In some embodiments, the humanized anti-TfR receptor antibodiesdescribed herein can be in any antibody form, including, but not limitedto, intact (i.e., full-length) antibodies, antigen-binding fragmentsthereof (such as Fab, Fab′, F(ab′)2, Fv), single chain antibodies,bi-specific antibodies, or nanobodies. In some embodiments, humanizedthe anti-TfR antibody described herein is a scFv. In some embodiments,the humanized anti-TfR antibody described herein is a scFv-Fab (e.g.,scFv fused to a portion of a constant region). In some embodiments, theanti-TfR receptor antibody described herein is a scFv fused to aconstant region (e.g., human IgG1 constant region as set forth in SEQ IDNO: 81 or SEQ ID NO: 82, or a portion thereof such as the Fc portion) ateither the N-terminus of C-terminus.

In some embodiments, conservative mutations can be introduced intoantibody sequences (e.g., CDRs or framework sequences) at positionswhere the residues are not likely to be involved in interacting with atarget antigen (e.g., transferrin receptor), for example, as determinedbased on a crystal structure. In some embodiments, one, two or moremutations (e.g., amino acid substitutions) are introduced into the Fcregion of an anti-TfR antibody described herein (e.g., in a CH2 domain(residues 231-340 of human IgG1) and/or (e.g., and) CH3 domain (residues341-447 of human IgG1) and/or (e.g., and) the hinge region, withnumbering according to the Kabat numbering system (e.g., the EU index inKabat)) to alter one or more functional properties of the antibody, suchas serum half-life, complement fixation, Fc receptor binding and/or(e.g., and) antigen-dependent cellular cytotoxicity.

In some embodiments, one, two or more mutations (e.g., amino acidsubstitutions) are introduced into the hinge region of the Fc region(CH1 domain) such that the number of cysteine residues in the hingeregion are altered (e.g., increased or decreased) as described in, e.g.,U.S. Pat. No. 5,677,425. The number of cysteine residues in the hingeregion of the CH1 domain can be altered to, e.g., facilitate assembly ofthe light and heavy chains, or to alter (e.g., increase or decrease) thestability of the antibody or to facilitate linker conjugation.

In some embodiments, one, two or more mutations (e.g., amino acidsubstitutions) are introduced into the Fc region of a muscle-targetingantibody described herein (e.g., in a CH2 domain (residues 231-340 ofhuman IgG1) and/or (e.g., and) CH3 domain (residues 341-447 of humanIgG1) and/or (e.g., and) the hinge region, with numbering according tothe Kabat numbering system (e.g., the EU index in Kabat)) to increase ordecrease the affinity of the antibody for an Fc receptor (e.g., anactivated Fc receptor) on the surface of an effector cell. Mutations inthe Fc region of an antibody that decrease or increase the affinity ofan antibody for an Fc receptor and techniques for introducing suchmutations into the Fc receptor or fragment thereof are known to one ofskill in the art. Examples of mutations in the Fc receptor of anantibody that can be made to alter the affinity of the antibody for anFc receptor are described in, e.g., Smith P et al., (2012) PNAS 109:6181-6186, U.S. Pat. No. 6,737,056, and International Publication Nos.WO 02/060919; WO 98/23289; and WO 97/34631, which are incorporatedherein by reference.

In some embodiments, one, two or more amino acid mutations (i.e.,substitutions, insertions or deletions) are introduced into an IgGconstant domain, or FcRn-binding fragment thereof (preferably an Fc orhinge-Fc domain fragment) to alter (e.g., decrease or increase)half-life of the antibody in vivo. See, e.g., International PublicationNos. WO 02/060919; WO 98/23289; and WO 97/34631; and U.S. Pat. Nos.5,869,046, 6,121,022, 6,277,375 and 6,165,745 for examples of mutationsthat will alter (e.g., decrease or increase) the half-life of anantibody in vivo.

In some embodiments, one, two or more amino acid mutations (i.e.,substitutions, insertions or deletions) are introduced into an IgGconstant domain, or FcRn-binding fragment thereof (preferably an Fc orhinge-Fc domain fragment) to decrease the half-life of the anti-anti-TfRantibody in vivo. In some embodiments, one, two or more amino acidmutations (i.e., substitutions, insertions or deletions) are introducedinto an IgG constant domain, or FcRn-binding fragment thereof(preferably an Fc or hinge-Fc domain fragment) to increase the half-lifeof the antibody in vivo. In some embodiments, the antibodies can haveone or more amino acid mutations (e.g., substitutions) in the secondconstant (CH2) domain (residues 231-340 of human IgG1) and/or (e.g.,and) the third constant (CH3) domain (residues 341-447 of human IgG1),with numbering according to the EU index in Kabat (Kabat E A et al.,(1991) supra). In some embodiments, the constant region of the IgG1 ofan antibody described herein comprises a methionine (M) to tyrosine (Y)substitution in position 252, a serine (S) to threonine (T) substitutionin position 254, and a threonine (T) to glutamic acid (E) substitutionin position 256, numbered according to the EU index as in Kabat. SeeU.S. Pat. No. 7,658,921, which is incorporated herein by reference. Thistype of mutant IgG, referred to as “YTE mutant” has been shown todisplay fourfold increased half-life as compared to wild-type versionsof the same antibody (see Dall'Acqua W F et al., (2006) J Biol Chem 281:23514-24). In some embodiments, an antibody comprises an IgG constantdomain comprising one, two, three or more amino acid substitutions ofamino acid residues at positions 251-257, 285-290, 308-314, 385-389, and428-436, numbered according to the EU index as in Kabat.

In some embodiments, one, two or more amino acid substitutions areintroduced into an IgG constant domain Fc region to alter the effectorfunction(s) of the anti-anti-TfR antibody. The effector ligand to whichaffinity is altered can be, for example, an Fc receptor or the Clcomponent of complement. This approach is described in further detail inU.S. Pat. Nos. 5,624,821 and 5,648,260. In some embodiments, thedeletion or inactivation (through point mutations or other means) of aconstant region domain can reduce Fc receptor binding of the circulatingantibody thereby increasing tumor localization. See, e.g., U.S. Pat.Nos. 5,585,097 and 8,591,886 for a description of mutations that deleteor inactivate the constant domain and thereby increase tumorlocalization. In some embodiments, one or more amino acid substitutionsmay be introduced into the Fc region of an antibody described herein toremove potential glycosylation sites on Fc region, which may reduce Fcreceptor binding (see, e.g., Shields R L et al., (2001) J Biol Chem 276:6591-604).

In some embodiments, one or more amino in the constant region of ananti-TfR antibody described herein can be replaced with a differentamino acid residue such that the antibody has altered C1q binding and/or(e.g., and) reduced or abolished complement dependent cytotoxicity(CDC). This approach is described in further detail in U.S. Pat. No.6,194,551 (Idusogie et al). In some embodiments, one or more amino acidresidues in the N-terminal region of the CH2 domain of an antibodydescribed herein are altered to thereby alter the ability of theantibody to fix complement. This approach is described further inInternational Publication No. WO 94/29351. In some embodiments, the Fcregion of an antibody described herein is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or (e.g., and) to increase the affinity of theantibody for an Fcγ receptor. This approach is described further inInternational Publication No. WO 00/42072.

In some embodiments, the heavy and/or (e.g., and) light chain variabledomain(s) sequence(s) of the antibodies provided herein can be used togenerate, for example, CDR-grafted, chimeric, humanized, or compositehuman antibodies or antigen-binding fragments, as described elsewhereherein. As understood by one of ordinary skill in the art, any variant,CDR-grafted, chimeric, humanized, or composite antibodies derived fromany of the antibodies provided herein may be useful in the compositionsand methods described herein and will maintain the ability tospecifically bind transferrin receptor, such that the variant,CDR-grafted, chimeric, humanized, or composite antibody has at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95% or more binding to transferrin receptor relative to the originalantibody from which it is derived.

In some embodiments, the antibodies provided herein comprise mutationsthat confer desirable properties to the antibodies. For example, toavoid potential complications due to Fab-arm exchange, which is known tooccur with native IgG4 mAbs, the antibodies provided herein may comprisea stabilizing ‘Adair’ mutation (Angal S., et al., “A single amino acidsubstitution abolishes the heterogeneity of chimeric mouse/human (IgG4)antibody,” Mol Immunol 30, 105-108; 1993), where serine 228 (EUnumbering; residue 241 Kabat numbering) is converted to prolineresulting in an IgG1-like hinge sequence. Accordingly, any of theantibodies may include a stabilizing ‘Adair’ mutation.

In some embodiments, an antibody is modified, e.g., modified viaglycosylation, phosphorylation, sumoylation, and/or (e.g., and)methylation. In some embodiments, an antibody is a glycosylatedantibody, which is conjugated to one or more sugar or carbohydratemolecules. In some embodiments, the one or more sugar or carbohydratemolecule are conjugated to the antibody via N-glycosylation,O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment),and/or (e.g., and) phosphoglycosylation. In some embodiments, the one ormore sugar or carbohydrate molecules are monosaccharides, disaccharides,oligosaccharides, or glycans. In some embodiments, the one or more sugaror carbohydrate molecule is a branched oligosaccharide or a branchedglycan. In some embodiments, the one or more sugar or carbohydratemolecule includes a mannose unit, a glucose unit, an N-acetylglucosamineunit, an N-acetylgalactosamine unit, a galactose unit, a fucose unit, ora phospholipid unit. In some embodiments, there are about 1-10, about1-5, about 5-10, about 1-4, about 1-3, or about 2 sugar molecules. Insome embodiments, a glycosylated antibody is fully or partiallyglycosylated. In some embodiments, an antibody is glycosylated bychemical reactions or by enzymatic means. In some embodiments, anantibody is glycosylated in vitro or inside a cell, which may optionallybe deficient in an enzyme in the N- or O-glycosylation pathway, e.g. aglycosyltransferase. In some embodiments, an antibody is functionalizedwith sugar or carbohydrate molecules as described in InternationalPatent Application Publication WO2014065661, published on May 1, 2014,entitled, “Modified antibody, antibody-conjugate and process for thepreparation thereof”.

In some embodiments, any one of the anti-TfR1 antibodies describedherein may comprise a signal peptide in the heavy and/or (e.g., and)light chain sequence (e.g., a N-terminal signal peptide). In someembodiments, the anti-TfR1 antibody described herein comprises any oneof the VH and VL sequences, any one of the IgG heavy chain and lightchain sequences, or any one of the Fab heavy chain and light chainsequences described herein, and further comprises a signal peptide(e.g., a N-terminal signal peptide). In some embodiments, the signalpeptide comprises the amino acid sequence of MGWSCIILFLVATATGVHS (SEQ IDNO: 104).

Other Known Anti-Transferrin Receptor Antibodies

Any other appropriate anti-transferrin receptor antibodies known in theart may be used as the muscle-targeting agent in the complexes disclosedherein. Examples of known anti-transferrin receptor antibodies,including associated references and binding epitopes, are listed inTable 6. In some embodiments, the anti-transferrin receptor antibodycomprises the complementarity determining regions (CDR-H1, CDR-H2,CDR-H3, CDR-L1, CDR-L2, and CDR-L3) of any of the anti-transferrinreceptor antibodies provided herein, e.g., anti-transferrin receptorantibodies listed in Table 6.

TABLE 6 List of anti-transferrin receptor antibody clones, includingassociated references and binding epitope information. Antibody CloneName Reference(s) Epitope/Notes OKT9 U.S. Pat. No. 4,364,934, filed Dec.4, 1979, Apical domain of TfR entitled “MONOCLONAL ANTIBODY (residues305-366 of TO A HUMAN EARLY THYMOCYTE human TfR sequence ANTIGEN ANDMETHODS FOR XM_052730.3, PREPARING SAME” available in GenBank) SchneiderC. et al. “Structural features of the cell surface receptor fortransferrin that is recognized by the monoclonal antibody OKT9.” J BiolChem. 1982, 257: 14, 8516-8522. (From JCR) WO 2015/098989, filed Dec.24, Apical domain Clone M11 Clone M23 2014, “Novel anti-Transferrinreceptor (residues 230-244 Clone M27 Clone B84 antibody that passesthrough and 326-347 of TfR) blood-brain barrier” and protease-like U.S.Pat. No. 9,994,641, filed Dec. 24, domain (residues 2014, “Novelanti-Transferrin receptor 461-473) antibody that passes throughblood-brain barrier” (From Genentech) WO 2016/081643, filed May 26,2016, Apical domain and 7A4, 8A2, 15D2, 10D11, entitled“ANTI-TRANSFERRIN non-apical regions 7B10, 15G11, 16G5, RECEPTORANTIBODIES AND 13C3, 16G4, 16F6, 7G7, METHODS OF USE” 4C2, 1B12, and13D4 U.S. Pat. No. 9,708,406, filed May 20, 2014, “Anti-transferrinreceptor antibodies and methods of use” (From Armagen) Lee et al.“Targeting Rat Anti-Mouse 8D3 Transferrin Receptor Monoclonal Antibodiesthrough Blood-Brain Barrier in Mouse” 2000, J Pharmacol. Exp. Ther.,292: 1048-1052. US Patent App. 2010/077498, filed Sep. 11, 2008,entitled “COMPOSITIONS AND METHODS FOR BLOOD-BRAIN BARRIER DELIVERY INTHE MOUSE” OX26 Haobam, B. et al. 2014. Rab17-mediated recyclingendosomes contribute to autophagosome formation in response to Group AStreptococcus invasion. Cellular microbiology. 16: 1806-21. DF1513Ortiz-Zapater E et al. Trafficking of the human transferrin receptor inplant cells: effects of tyrphostin A23 and brefeldin A. Plant J 48:757-70 (2006). 1A1B2, 66IG10, MEM-189, Commercially availableanti-transferrin Novus Biologicals JF0956, 29806, 1A1B2, receptorantibodies. 8100 Southpark Way, TFRC/1818, 1E6, 66Ig10, A-8 Littleton COTFRC/1059, Q1/71, 23D10, 80120 13E4, TFRC/1149, ER-MP21, YTA74.4, BU54,2B6, RI7 217 (From INSERM) US Patent App. 2011/0311544A1, filed Does notcompete BA120g Jun. 15, 2005, entitled “ANTI-CD71 with OKT9 MONOCLONALANTIBODIES AND USES THEREOF FOR TREATING MALIGNANT TUMOR CELLS” LUCA31U.S. Pat. No. 7,572,895, filed Jun. 7, 2004, “LUCA31 epitope” entitled“TRANSFERRIN RECEPTOR ANTIBODIES” (Salk Institute) Trowbridge, I. S. etal. “Anti-transferrin B3/25 T58/30 receptor monoclonal antibody andtoxin-antibody conjugates affect growth of human tumour cells.” Nature,1981, volume 294, pages 171-173 R17 217.1.3, 5E9C11, Commerciallyavailable anti-transferrin BioXcell 10 Technology OKT9 (BE0023 clone)receptor antibodies. Dr., Suite 2B West Lebanon, NH 03784-1671 USABK19.9, B3/25, T56/14 Gatter, K. C. et al. “Transferrin receptors andT58/1 in human tissues: their distribution and possible clinicalrelevance.” J Clin Pathol. 1983 May; 36 (5): 539-45.

In some embodiments, transferrin receptor antibodies of the presentdisclosure include one or more of the CDR-H (e.g., CDR-H1, CDR-H2, andCDR-H3) amino acid sequences from any one of the anti-transferrinreceptor antibodies selected from Table 6. In some embodiments,transferrin receptor antibodies include the CDR-H1, CDR-H2, and CDR-H3as provided for any one of the anti-transferrin receptor antibodiesselected from Table 6. In some embodiments, anti-transferrin receptorantibodies include the CDR-L1, CDR-L2, and CDR-L3 as provided for anyone of the anti-transferrin receptor antibodies selected from Table 6.In some embodiments, anti-transferrin antibodies include the CDR-H1,CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 as provided for any one ofthe anti-transferrin receptor antibodies selected from Table 6. Thedisclosure also includes any nucleic acid sequence that encodes amolecule comprising a CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, or CDR-L3as provided for any one of the anti-transferrin receptor antibodiesselected from Table 6. In some embodiments, antibody heavy and lightchain CDR3 domains may play a particularly important role in the bindingspecificity/affinity of an antibody for an antigen. Accordingly,anti-transferrin receptor antibodies of the disclosure may include atleast the heavy and/or (e.g., and) light chain CDR3s of any one of theanti-transferrin receptor antibodies selected from Table 6.

In some examples, any of the anti-transferrin receptor antibodies of thedisclosure have one or more CDR (e.g., CDR-H or CDR-L) sequencessubstantially similar to any of the CDR-H1, CDR-H2, CDR-H3, CDR-L1,CDR-L2, and/or (e.g., and) CDR-L3 sequences from one of theanti-transferrin receptor antibodies selected from Table 6. In someembodiments, the position of one or more CDRs along the VH (e.g.,CDR-H1, CDR-H2, or CDR-H3) and/or (e.g., and) VL (e.g., CDR-L1, CDR-L2,or CDR-L3) region of an antibody described herein can vary by one, two,three, four, five, or six amino acid positions so long as immunospecificbinding to transferrin receptor (e.g., human transferrin receptor) ismaintained (e.g., substantially maintained, for example, at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, at least 95% ofthe binding of the original antibody from which it is derived). Forexample, in some embodiments, the position defining a CDR of anyantibody described herein can vary by shifting the N-terminal and/or(e.g., and) C-terminal boundary of the CDR by one, two, three, four,five, or six amino acids, relative to the CDR position of any one of theantibodies described herein, so long as immunospecific binding totransferrin receptor (e.g., human transferrin receptor) is maintained(e.g., substantially maintained, for example, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95% of thebinding of the original antibody from which it is derived). In anotherembodiment, the length of one or more CDRs along the VH (e.g., CDR-H1,CDR-H2, or CDR-H3) and/or (e.g., and) VL (e.g., CDR-L1, CDR-L2, orCDR-L3) region of an antibody described herein can vary (e.g., beshorter or longer) by one, two, three, four, five, or more amino acids,so long as immunospecific binding to transferrin receptor (e.g., humantransferrin receptor) is maintained (e.g., substantially maintained, forexample, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95% of the binding of the original antibody fromwhich it is derived).

Accordingly, in some embodiments, a CDR-L1, CDR-L2, CDR-L3, CDR-H1,CDR-H2, and/or (e.g., and) CDR-H3 described herein may be one, two,three, four, five or more amino acids shorter than one or more of theCDRs described herein (e.g., CDRs from any of the anti-transferrinreceptor antibodies selected from Table 6) so long as immunospecificbinding to transferrin receptor (e.g., human transferrin receptor) ismaintained (e.g., substantially maintained, for example, at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, at least 95%relative to the binding of the original antibody from which it isderived). In some embodiments, a CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2,and/or (e.g., and) CDR-H3 described herein may be one, two, three, four,five or more amino acids longer than one or more of the CDRs describedherein (e.g., CDRs from any of the anti-transferrin receptor antibodiesselected from Table 6) so long as immunospecific binding to transferrinreceptor (e.g., human transferrin receptor) is maintained (e.g.,substantially maintained, for example, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95% relative to thebinding of the original antibody from which it is derived). In someembodiments, the amino portion of a CDR-L1, CDR-L2, CDR-L3, CDR-H1,CDR-H2, and/or (e.g., and) CDR-H3 described herein can be extended byone, two, three, four, five or more amino acids compared to one or moreof the CDRs described herein (e.g., CDRs from any of theanti-transferrin receptor antibodies selected from Table 6) so long asimmunospecific binding to transferrin receptor (e.g., human transferrinreceptor is maintained (e.g., substantially maintained, for example, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95% relative to the binding of the original antibody from which itis derived). In some embodiments, the carboxy portion of a CDR-L1,CDR-L2, CDR-L3, CDR-H1, CDR-H2, and/or (e.g., and) CDR-H3 describedherein can be extended by one, two, three, four, five or more aminoacids compared to one or more of the CDRs described herein (e.g., CDRsfrom any of the anti-transferrin receptor antibodies selected from Table6) so long as immunospecific binding to transferrin receptor (e.g.,human transferrin receptor) is maintained (e.g., substantiallymaintained, for example, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95% relative to the binding of theoriginal antibody from which it is derived). In some embodiments, theamino portion of a CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and/or (e.g.,and) CDR-H3 described herein can be shortened by one, two, three, four,five or more amino acids compared to one or more of the CDRs describedherein (e.g., CDRs from any of the anti-transferrin receptor antibodiesselected from Table 6) so long as immunospecific binding to transferrinreceptor (e.g., human transferrin receptor) is maintained (e.g.,substantially maintained, for example, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95% relative to thebinding of the original antibody from which it is derived). In someembodiments, the carboxy portion of a CDR-L1, CDR-L2, CDR-L3, CDR-H1,CDR-H2, and/or (e.g., and) CDR-H3 described herein can be shortened byone, two, three, four, five or more amino acids compared to one or moreof the CDRs described herein (e.g., CDRs from any of theanti-transferrin receptor antibodies selected from Table 6) so long asimmunospecific binding to transferrin receptor (e.g., human transferrinreceptor) is maintained (e.g., substantially maintained, for example, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95% relative to the binding of the original antibody from which itis derived). Any method can be used to ascertain whether immunospecificbinding to transferrin receptor (e.g., human transferrin receptor) ismaintained, for example, using binding assays and conditions describedin the art.

In some examples, any of the anti-transferrin receptor antibodies of thedisclosure have one or more CDR (e.g., CDR-H or CDR-L) sequencessubstantially similar to any one of the anti-transferrin receptorantibodies selected from Table 6. For example, the antibodies mayinclude one or more CDR sequence(s) from any of the anti-transferrinreceptor antibodies selected from Table 6 containing up to 5, 4, 3, 2,or 1 amino acid residue variations as compared to the corresponding CDRregion in any one of the CDRs provided herein (e.g., CDRs from any ofthe anti-transferrin receptor antibodies selected from Table 6) so longas immunospecific binding to transferrin receptor (e.g., humantransferrin receptor) is maintained (e.g., substantially maintained, forexample, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95% relative to the binding of the original antibodyfrom which it is derived). In some embodiments, any of the amino acidvariations in any of the CDRs provided herein may be conservativevariations. Conservative variations can be introduced into the CDRs atpositions where the residues are not likely to be involved ininteracting with a transferrin receptor protein (e.g., a humantransferrin receptor protein), for example, as determined based on acrystal structure. Some aspects of the disclosure provide transferrinreceptor antibodies that comprise one or more of the heavy chainvariable (VH) and/or (e.g., and) light chain variable (VL) domainsprovided herein. In some embodiments, any of the VH domains providedherein include one or more of the CDR-H sequences (e.g., CDR-H1, CDR-H2,and CDR-H3) provided herein, for example, any of the CDR-H sequencesprovided in any one of the anti-transferrin receptor antibodies selectedfrom Table 6. In some embodiments, any of the VL domains provided hereininclude one or more of the CDR-L sequences (e.g., CDR-L1, CDR-L2, andCDR-L3) provided herein, for example, any of the CDR-L sequencesprovided in any one of the anti-transferrin receptor antibodies selectedfrom Table 6.

In some embodiments, anti-transferrin receptor antibodies of thedisclosure include any antibody that includes a heavy chain variabledomain and/or (e.g., and) a light chain variable domain of anyanti-transferrin receptor antibody, such as any one of theanti-transferrin receptor antibodies selected from Table 6. In someembodiments, anti-transferrin receptor antibodies of the disclosureinclude any antibody that includes the heavy chain variable and lightchain variable pairs of any anti-transferrin receptor antibody, such asany one of the anti-transferrin receptor antibodies selected from Table6.

Aspects of the disclosure provide anti-transferrin receptor antibodieshaving a heavy chain variable (VH) and/or (e.g., and) a light chainvariable (VL) domain amino acid sequence homologous to any of thosedescribed herein. In some embodiments, the anti-transferrin receptorantibody comprises a heavy chain variable sequence or a light chainvariable sequence that is at least 75% (e.g., 80%, 85%, 90%, 95%, 98%,or 99%) identical to the heavy chain variable sequence and/or any lightchain variable sequence of any anti-transferrin receptor antibody, suchas any one of the anti-transferrin receptor antibodies selected fromTable 6. In some embodiments, the homologous heavy chain variable and/or(e.g., and) a light chain variable amino acid sequences do not varywithin any of the CDR sequences provided herein. For example, in someembodiments, the degree of sequence variation (e.g., 75%, 80%, 85%, 90%,95%, 98%, or 99%) may occur within a heavy chain variable and/or (e.g.,and) a light chain variable sequence excluding any of the CDR sequencesprovided herein. In some embodiments, any of the anti-transferrinreceptor antibodies provided herein comprise a heavy chain variablesequence and a light chain variable sequence that comprises a frameworksequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identicalto the framework sequence of any anti-transferrin receptor antibody,such as any one of the anti-transferrin receptor antibodies selectedfrom Table 6.

In some embodiments, an anti-transferrin receptor antibody, whichspecifically binds to transferrin receptor (e.g., human transferrinreceptor), comprises a light chain variable VL domain comprising any ofthe CDR-L domains (CDR-L1, CDR-L2, and CDR-L3), or CDR-L domain variantsprovided herein, of any of the anti-transferrin receptor antibodiesselected from Table 6. In some embodiments, an anti-transferrin receptorantibody, which specifically binds to transferrin receptor (e.g., humantransferrin receptor), comprises a light chain variable VL domaincomprising the CDR-L1, the CDR-L2, and the CDR-L3 of anyanti-transferrin receptor antibody, such as any one of theanti-transferrin receptor antibodies selected from Table 6. In someembodiments, the anti-transferrin receptor antibody comprises a lightchain variable (VL) region sequence comprising one, two, three or fourof the framework regions of the light chain variable region sequence ofany anti-transferrin receptor antibody, such as any one of theanti-transferrin receptor antibodies selected from Table 6. In someembodiments, the anti-transferrin receptor antibody comprises one, two,three or four of the framework regions of a light chain variable regionsequence which is at least 75%, 80%, 85%, 90%, 95%, or 100% identical toone, two, three or four of the framework regions of the light chainvariable region sequence of any anti-transferrin receptor antibody, suchas any one of the anti-transferrin receptor antibodies selected fromTable 6. In some embodiments, the light chain variable framework regionthat is derived from said amino acid sequence consists of said aminoacid sequence but for the presence of up to 10 amino acid substitutions,deletions, and/or (e.g., and) insertions, preferably up to 10 amino acidsubstitutions. In some embodiments, the light chain variable frameworkregion that is derived from said amino acid sequence consists of saidamino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidresidues being substituted for an amino acid found in an analogousposition in a corresponding non-human, primate, or human light chainvariable framework region.

In some embodiments, an anti-transferrin receptor antibody thatspecifically binds to transferrin receptor comprises the CDR-L1, theCDR-L2, and the CDR-L3 of any anti-transferrin receptor antibody, suchas any one of the anti-transferrin receptor antibodies selected fromTable 6. In some embodiments, the antibody further comprises one, two,three or all four VL framework regions derived from the VL of a human orprimate antibody. The primate or human light chain framework region ofthe antibody selected for use with the light chain CDR sequencesdescribed herein, can have, for example, at least 70% (e.g., at least75%, 80%, 85%, 90%, 95%, 98%, or at least 99%) identity with a lightchain framework region of a non-human parent antibody. The primate orhuman antibody selected can have the same or substantially the samenumber of amino acids in its light chain complementarity determiningregions to that of the light chain complementarity determining regionsof any of the antibodies provided herein, e.g., any of theanti-transferrin receptor antibodies selected from Table 6. In someembodiments, the primate or human light chain framework region aminoacid residues are from a natural primate or human antibody light chainframework region having at least 75% identity, at least 80% identity, atleast 85% identity, at least 90% identity, at least 95% identity, atleast 98% identity, at least 99% (or more) identity with the light chainframework regions of any anti-transferrin receptor antibody, such as anyone of the anti-transferrin receptor antibodies selected from Table 6.In some embodiments, an anti-transferrin receptor antibody furthercomprises one, two, three or all four VL framework regions derived froma human light chain variable kappa subfamily. In some embodiments, ananti-transferrin receptor antibody further comprises one, two, three orall four VL framework regions derived from a human light chain variablelambda subfamily.

In some embodiments, any of the anti-transferrin receptor antibodiesprovided herein comprise a light chain variable domain that furthercomprises a light chain constant region. In some embodiments, the lightchain constant region is a kappa, or a lambda light chain constantregion. In some embodiments, the kappa or lambda light chain constantregion is from a mammal, e.g., from a human, monkey, rat, or mouse. Insome embodiments, the light chain constant region is a human kappa lightchain constant region. In some embodiments, the light chain constantregion is a human lambda light chain constant region. It should beappreciated that any of the light chain constant regions provided hereinmay be variants of any of the light chain constant regions providedherein. In some embodiments, the light chain constant region comprisesan amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or99% identical to any of the light chain constant regions of anyanti-transferrin receptor antibody, such as any one of theanti-transferrin receptor antibodies selected from Table 6.

In some embodiments, the anti-transferrin receptor antibody is anyanti-transferrin receptor antibody, such as any one of theanti-transferrin receptor antibodies selected from Table 6.

In some embodiments, an anti-transferrin receptor antibody comprises aVL domain comprising the amino acid sequence of any anti-transferrinreceptor antibody, such as any one of the anti-transferrin receptorantibodies selected from Table 6, and wherein the constant regionscomprise the amino acid sequences of the constant regions of an IgG,IgE, IgM, IgD, IgA or IgY immunoglobulin molecule, or a human IgG, IgE,IgM, IgD, IgA or IgY immunoglobulin molecule. In some embodiments, ananti-transferrin receptor antibody comprises any of the VL domains, orVL domain variants, and any of the VH domains, or VH domain variants,wherein the VL and VH domains, or variants thereof, are from the sameantibody clone, and wherein the constant regions comprise the amino acidsequences of the constant regions of an IgG, IgE, IgM, IgD, IgA or IgYimmunoglobulin molecule, any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulinmolecule. Non-limiting examples of human constant regions are describedin the art, e.g., see Kabat E A et al., (1991) supra.

In some embodiments, the muscle-targeting agent is a transferrinreceptor antibody (e.g., the antibody and variants thereof as describedin International Application Publication WO 2016/081643, incorporatedherein by reference).

The heavy chain and light chain CDRs of the antibody according todifferent definition systems are provided in Table 7. The differentdefinition systems, e.g., the Kabat definition, the Chothia definition,and/or (e.g., and) the contact definition have been described. See,e.g., (e.g., Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242, Chothia et al., (1989)Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917,Al-lazikani et al (1997) J. Molec. Biol. 273:927-948; and Almagro, J.Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk andbioinf.org.uk/abs).

TABLE 7Heavy chain and light chain CDRs of a mouse transferrin receptor antibodyCDRs Kabat Chothia Contact CDR-H1 SYWMH (SEQ ID NO: GYTFTSY (SEQ ID NO:TSYWMH (SEQ ID NO: 110) 116) 118) CDR-H2 EINPTNGRTNYIEKFKSNPTNGR (SEQ ID NO: WIGEINPTNGRTN (SEQ ID NO: 111) 117) (SEQ ID NO: 119)CDR-H3 GTRAYHY (SEQ ID GTRAYHY (SEQ ID ARGTRA (SEQ ID NO: NO: 112)NO: 112) 120) CDR-L1 RASDNLYSNLA (SEQ RASDNLYSNLA (SEQ YSNLAWY (SEQ IDID NO: 113) ID NO: 113) NO: 121) CDR-L2 DATNLAD (SEQ ID NO:DATNLAD (SEQ ID LLVYDATNLA (SEQ ID 114) NO: 114) NO: 122) CDR-L3QHFWGTPLT (SEQ ID QHFWGTPLT (SEQ ID QHFWGTPL (SEQ ID NO: 115) NO: 115)NO: 123)

The heavy chain variable domain (VH) and light chain variable domainsequences are also provided:

VH (SEQ ID NO: 124) QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPTNGRTNYIEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYC ARGTRAYHYWGQGTSVTVSS VL(SEQ ID NO: 125) DIQMTQSPASLSVSVGETVTITCRASDNLYSNLAWYQQKQGKSPQLLVYDATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWGTPL  TFGAGTKLELK

In some embodiments, the transferrin receptor antibody of the presentdisclosure comprises a CDR-H1, a CDR-H2, and a CDR-H3 that are the sameas the CDR-H1, CDR-H2, and CDR-H3 shown in Table 7. Alternatively or inaddition (e.g., in addition), the transferrin receptor antibody of thepresent disclosure comprises a CDR-L1, a CDR-L2, and a CDR-L3 that arethe same as the CDR-L1, CDR-L2, and CDR-L3 shown in Table 7.

In some embodiments, the transferrin receptor antibody of the presentdisclosure comprises a CDR-H1, a CDR-H2, and a CDR-H3, whichcollectively contains no more than 5 amino acid variations (e.g., nomore than 5, 4, 3, 2, or 1 amino acid variation) as compared with theCDR-H1, CDR-H2, and CDR-H3 as shown in Table 7. “Collectively” meansthat the total number of amino acid variations in all of the three heavychain CDRs is within the defined range. Alternatively or in addition(e.g., in addition), the transferrin receptor antibody of the presentdisclosure may comprise a CDR-L1, a CDR-L2, and a CDR-L3, whichcollectively contains no more than 5 amino acid variations (e.g., nomore than 5, 4, 3, 2 or 1 amino acid variation) as compared with theCDR-L1, CDR-L2, and CDR-L3 as shown in Table 7.

In some embodiments, the transferrin receptor antibody of the presentdisclosure comprises a CDR-H1, a CDR-H2, and a CDR-H3, at least one ofwhich contains no more than 3 amino acid variations (e.g., no more than3, 2, or 1 amino acid variation) as compared with the counterpart heavychain CDR as shown in Table 7. Alternatively or in addition (e.g., inaddition), the transferrin receptor antibody of the present disclosuremay comprise CDR-L1, a CDR-L2, and a CDR-L3, at least one of whichcontains no more than 3 amino acid variations (e.g., no more than 3, 2,or 1 amino acid variation) as compared with the counterpart light chainCDR as shown in Table 7.

In some embodiments, the transferrin receptor antibody of the presentdisclosure comprises a CDR-L3, which contains no more than 3 amino acidvariations (e.g., no more than 3, 2, or 1 amino acid variation) ascompared with the CDR-L3 as shown in Table 7. In some embodiments, thetransferrin receptor antibody of the present disclosure comprises aCDR-L3 containing one amino acid variation as compared with the CDR-L3as shown in Table 7. In some embodiments, the transferrin receptorantibody of the present disclosure comprises a CDR-L3 of QHFAGTPLT (SEQID NO: 126) according to the Kabat and Chothia definition system) orQHFAGTPL (SEQ ID NO: 127) according to the Contact definition system).In some embodiments, the transferrin receptor antibody of the presentdisclosure comprises a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1 and a CDR-L2that are the same as the CDR-H1, CDR-H2, and CDR-H3 shown in Table 7,and comprises a CDR-L3 of QHFAGTPLT (SEQ ID NO: 126) according to theKabat and Chothia definition system) or QHFAGTPL (SEQ ID NO: 127)according to the Contact definition system).

In some embodiments, the transferrin receptor antibody of the presentdisclosure comprises heavy chain CDRs that collectively are at least 80%(e.g., 80%, 85%, 90%, 95%, or 98%) identical to the heavy chain CDRs asshown in Table 7. Alternatively or in addition (e.g., in addition), thetransferrin receptor antibody of the present disclosure comprises lightchain CDRs that collectively are at least 80% (e.g., 80%, 85%, 90%, 95%,or 98%) identical to the light chain CDRs as shown in Table 7.

In some embodiments, the transferrin receptor antibody of the presentdisclosure comprises a VH comprising the amino acid sequence of SEQ IDNO: 124. Alternatively or in addition (e.g., in addition), thetransferrin receptor antibody of the present disclosure comprises a VLcomprising the amino acid sequence of SEQ ID NO: 125.

In some embodiments, the transferrin receptor antibody of the presentdisclosure comprises a VH containing no more than 25 amino acidvariations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) as compared with the VH as set forth in SEQ ID NO: 124.Alternatively or in addition (e.g., in addition), the transferrinreceptor antibody of the present disclosure comprises a VL containing nomore than 15 amino acid variations (e.g., no more than 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) as compared with the VL as set forth in SEQ ID NO: 125.

In some embodiments, the transferrin receptor antibody of the presentdisclosure comprises a VH comprising an amino acid sequence that is atleast 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the VH as setforth in SEQ ID NO: 124. Alternatively or in addition (e.g., inaddition), the transferrin receptor antibody of the present disclosurecomprises a VL comprising an amino acid sequence that is at least 80%(e.g., 80%, 85%, 90%, 95%, or 98%) identical to the VL as set forth inSEQ ID NO: 125.

In some embodiments, the transferrin receptor antibody of the presentdisclosure is a humanized antibody (e.g., a humanized variant of anantibody). In some embodiments, the transferrin receptor antibody of thepresent disclosure comprises a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, aCDR-L2, and a CDR-L3 that are the same as the CDR-H1, CDR-H2, and CDR-H3shown in Table 7, and comprises a humanized heavy chain variable regionand/or (e.g., and) a humanized light chain variable region.

Humanized antibodies are human immunoglobulins (recipient antibody) inwhich residues from a complementary determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat, or rabbit having the desiredspecificity, affinity, and capacity. In some embodiments, Fv frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, the humanized antibodymay comprise residues that are found neither in the recipient antibodynor in the imported CDR or framework sequences, but are included tofurther refine and optimize antibody performance. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin consensus sequence. The humanized antibody optimally alsowill comprise at least a portion of an immunoglobulin constant region ordomain (Fc), typically that of a human immunoglobulin. Antibodies mayhave Fc regions modified as described in WO 99/58572. Other forms ofhumanized antibodies have one or more CDRs (one, two, three, four, five,six) which are altered with respect to the original antibody, which arealso termed one or more CDRs derived from one or more CDRs from theoriginal antibody. Humanized antibodies may also involve affinitymaturation.

In some embodiments, humanization is achieved by grafting the CDRs(e.g., as shown in Table 7) into the IGKV1-NL1*01 and IGHV1-3*01 humanvariable domains. In some embodiments, the transferrin receptor antibodyof the present disclosure is a humanized variant comprising one or moreamino acid substitutions at positions 9, 13, 17, 18, 40, 45, and 70 ascompared with the VL as set forth in SEQ ID NO: 125, and/or (e.g., and)one or more amino acid substitutions at positions 1, 5, 7, 11, 12, 20,38, 40, 44, 66, 75, 81, 83, 87, and 108 as compared with the VH as setforth in SEQ ID NO: 124. In some embodiments, the transferrin receptorantibody of the present disclosure is a humanized variant comprisingamino acid substitutions at all of positions 9, 13, 17, 18, 40, 45, and70 as compared with the VL as set forth in SEQ ID NO: 125, and/or (e.g.,and) amino acid substitutions at all of positions 1, 5, 7, 11, 12, 20,38, 40, 44, 66, 75, 81, 83, 87, and 108 as compared with the VH as setforth in SEQ ID NO: 124.

In some embodiments, the transferrin receptor antibody of the presentdisclosure is a humanized antibody and contains the residues atpositions 43 and 48 of the VL as set forth in SEQ ID NO: 125.Alternatively or in addition (e.g., in addition), the transferrinreceptor antibody of the present disclosure is a humanized antibody andcontains the residues at positions 48, 67, 69, 71, and 73 of the VH asset forth in SEQ ID NO: 124.

The VH and VL amino acid sequences of an example humanized antibody thatmay be used in accordance with the present disclosure are provided:

Humanized VH (SEQ ID NO: 128)EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWIGEINPTNGRTNYIEKFKSRATLTVDKSASTAYMELSSLRSEDTAVYYCAR GTRAYHYWGQGTMVTVSSHumanized VL (SEQ ID NO: 129)DIQMTQSPSSLSASVGDRVTITCRASDNLYSNLAWYQQKPGKSPKLLVYDATNLADGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPLTF GQGTKVEIK

In some embodiments, the transferrin receptor antibody of the presentdisclosure comprises a VH comprising the amino acid sequence of SEQ IDNO: 128. Alternatively or in addition (e.g., in addition), thetransferrin receptor antibody of the present disclosure comprises a VLcomprising the amino acid sequence of SEQ ID NO: 129.

In some embodiments, the transferrin receptor antibody of the presentdisclosure comprises a VH containing no more than 25 amino acidvariations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) as compared with the VH as set forth in SEQ ID NO: 128.Alternatively or in addition (e.g., in addition), the transferrinreceptor antibody of the present disclosure comprises a VL containing nomore than 15 amino acid variations (e.g., no more than 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) as compared with the VL as set forth in SEQ ID NO: 129.

In some embodiments, the transferrin receptor antibody of the presentdisclosure comprises a VH comprising an amino acid sequence that is atleast 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the VH as setforth in SEQ ID NO: 128. Alternatively or in addition (e.g., inaddition), the transferrin receptor antibody of the present disclosurecomprises a VL comprising an amino acid sequence that is at least 80%(e.g., 80%, 85%, 90%, 95%, or 98%) identical to the VL as set forth inSEQ ID NO: 129.

In some embodiments, the transferrin receptor antibody of the presentdisclosure is a humanized variant comprising amino acid substitutions atone or more of positions 43 and 48 as compared with the VL as set forthin SEQ ID NO: 125, and/or (e.g., and) amino acid substitutions at one ormore of positions 48, 67, 69, 71, and 73 as compared with the VH as setforth in SEQ ID NO: 124. In some embodiments, the transferrin receptorantibody of the present disclosure is a humanized variant comprising aS43A and/or (e.g., and) a V48L mutation as compared with the VL as setforth in SEQ ID NO: 125, and/or (e.g., and) one or more of A67V, L69I,V71R, and K73T mutations as compared with the VH as set forth in SEQ IDNO: 124.

In some embodiments, the transferrin receptor antibody of the presentdisclosure is a humanized variant comprising amino acid substitutions atone or more of positions 9, 13, 17, 18, 40, 43, 48, 45, and 70 ascompared with the VL as set forth in SEQ ID NO: 125, and/or (e.g., and)amino acid substitutions at one or more of positions 1, 5, 7, 11, 12,20, 38, 40, 44, 48, 66, 67, 69, 71, 73, 75, 81, 83, 87, and 108 ascompared with the VH as set forth in SEQ ID NO: 124.

In some embodiments, the transferrin receptor antibody of the presentdisclosure is a chimeric antibody, which can include a heavy constantregion and a light constant region from a human antibody. Chimericantibodies refer to antibodies having a variable region or part ofvariable region from a first species and a constant region from a secondspecies. Typically, in these chimeric antibodies, the variable region ofboth light and heavy chains mimics the variable regions of antibodiesderived from one species of mammals (e.g., a non-human mammal such asmouse, rabbit, and rat), while the constant portions are homologous tothe sequences in antibodies derived from another mammal such as human.In some embodiments, amino acid modifications can be made in thevariable region and/or (e.g., and) the constant region.

In some embodiments, the transferrin receptor antibody described hereinis a chimeric antibody, which can include a heavy constant region and alight constant region from a human antibody. Chimeric antibodies referto antibodies having a variable region or part of variable region from afirst species and a constant region from a second species. Typically, inthese chimeric antibodies, the variable region of both light and heavychains mimics the variable regions of antibodies derived from onespecies of mammals (e.g., a non-human mammal such as mouse, rabbit, andrat), while the constant portions are homologous to the sequences inantibodies derived from another mammal such as human. In someembodiments, amino acid modifications can be made in the variable regionand/or (e.g., and) the constant region.

In some embodiments, the heavy chain of any of the transferrin receptorantibodies as described herein may comprises a heavy chain constantregion (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combinationthereof). The heavy chain constant region can of any suitable origin,e.g., human, mouse, rat, or rabbit. In one specific example, the heavychain constant region is from a human IgG (a gamma heavy chain), e.g.,IgG1, IgG2, or IgG4. An example of human IgG1 constant region is givenbelow:

(SEQ ID NO: 130) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 

In some embodiments, the light chain of any of the transferrin receptorantibodies described herein may further comprise a light chain constantregion (CL), which can be any CL known in the art. In some examples, theCL is a kappa light chain. In other examples, the CL is a lambda lightchain. In some embodiments, the CL is a kappa light chain, the sequenceof which is provided below:

(SEQ ID NO: 83) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV  TKSFNRGEC

Other antibody heavy and light chain constant regions are well known inthe art, e.g., those provided in the IMGT database (www.imgt.org) or atwww.vbase2.org/vbstat.php., both of which are incorporated by referenceherein.

Examples of heavy chain and light chain amino acid sequences of thetransferrin receptor antibodies described are provided below:

Heavy Chain (VH + human IgG1 constant region) (SEQ ID NO: 132)QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPTNGRTNYIEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGTRAYHYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGKLight Chain (VL + kappa light chain) (SEQ ID NO: 133)DIQMTQSPASLSVSVGETVTITCRASDNLYSNLAWYQQKQGKSPQLLVYDATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWGTPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECHeavy Chain (humanized VH + human IgG1 constant region) (SEQ ID NO: 134)EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWIGEINPTNGRTNYIEKFKSRATLTVDKSASTAYMELSSLRSEDTAVYYCARGTRAYHYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGKLight Chain (humanized VL + kappa light chain) (SEQ ID NO: 135)DIQMTQSPSSLSASVGDRVTITCRASDNLYSNLAWYQQKPGKSPKLLVYDATNLADGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC

In some embodiments, the transferrin receptor antibody described hereincomprises a heavy chain comprising an amino acid sequence that is atleast 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO:132. Alternatively or in addition (e.g., in addition), the transferrinreceptor antibody described herein comprises a light chain comprising anamino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, or98%) identical to SEQ ID NO: 133. In some embodiments, the transferrinreceptor antibody described herein comprises a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 132. Alternatively or in addition(e.g., in addition), the transferrin receptor antibody described hereincomprises a light chain comprising the amino acid sequence of SEQ ID NO:133.

In some embodiments, the transferrin receptor antibody of the presentdisclosure comprises a heavy chain containing no more than 25 amino acidvariations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) as compared with the heavy chain as set forth in SEQ ID NO:132. Alternatively or in addition (e.g., in addition), the transferrinreceptor antibody of the present disclosure comprises a light chaincontaining no more than 15 amino acid variations (e.g., no more than 20,19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 aminoacid variation) as compared with the light chain as set forth in SEQ IDNO: 133.

In some embodiments, the transferrin receptor antibody described hereincomprises a heavy chain comprising an amino acid sequence that is atleast 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO:134. Alternatively or in addition (e.g., in addition), the transferrinreceptor antibody described herein comprises a light chain comprising anamino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, or98%) identical to SEQ ID NO: 135. In some embodiments, the transferrinreceptor antibody described herein comprises a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 134. Alternatively or in addition(e.g., in addition), the transferrin receptor antibody described hereincomprises a light chain comprising the amino acid sequence of SEQ ID NO:135.

In some embodiments, the transferrin receptor antibody of the presentdisclosure comprises a heavy chain containing no more than 25 amino acidvariations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) as compared with the heavy chain of humanized antibody as setforth in SEQ ID NO: 134. Alternatively or in addition (e.g., inaddition), the transferrin receptor antibody of the present disclosurecomprises a light chain containing no more than 15 amino acid variations(e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6,5, 4, 3, 2, or 1 amino acid variation) as compared with the light chainof humanized antibody as set forth in SEQ ID NO: 135.

In some embodiments, the transferrin receptor antibody is an antigenbinding fragment (Fab) of an intact antibody (full-length antibody).Antigen binding fragment of an intact antibody (full-length antibody)can be prepared via routine methods. For example, F(ab′)2 fragments canbe produced by pepsin digestion of an antibody molecule, and Fab′fragments that can be generated by reducing the disulfide bridges ofF(ab′)2 fragments. Examples of Fab amino acid sequences of thetransferrin receptor antibodies described herein are provided below:

Heavy Chain Fab (VH + a portion of human IgG1 constant region)(SEQ ID NO: 136)QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPTNGRTNYIEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGTRAYHYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPHeavy Chain Fab (humanized VH + a portion of human IgG1 constant region)(SEQ ID NO: 137)EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWIGEINPTNGRTNYIEKFKSRATLTVDKSASTAYMELSSLRSEDTAVYYCARGTRAYHYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP

In some embodiments, the transferrin receptor antibody described hereincomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:136. Alternatively or in addition (e.g., in addition), the transferrinreceptor antibody described herein comprises a light chain comprisingthe amino acid sequence of SEQ ID NO: 133.

In some embodiments, the transferrin receptor antibody described hereincomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:137. Alternatively or in addition (e.g., in addition), the transferrinreceptor antibody described herein comprises a light chain comprisingthe amino acid sequence of SEQ ID NO: 135.

The transferrin receptor antibodies described herein can be in anyantibody form, including, but not limited to, intact (i.e., full-length)antibodies, antigen-binding fragments thereof (such as Fab, Fab′,F(ab′)2, Fv), single chain antibodies, bi-specific antibodies, ornanobodies. In some embodiments, the transferrin receptor antibodydescribed herein is a scFv. In some embodiments, the transferrinreceptor antibody described herein is a scFv-Fab (e.g., scFv fused to aportion of a constant region). In some embodiments, the transferrinreceptor antibody described herein is a scFv fused to a constant region(e.g., human IgG1 constant region as set forth in SEQ ID NO: 130).

In some embodiments, any one of the anti-TfR antibodies described hereinis produced by recombinant DNA technology in Chinese hamster ovary (CHO)cell suspension culture, optionally in CHO-K1 cell (e.g., CHO-K1 cellsderived from European Collection of Animal Cell Culture, Cat. No.85051005) suspension culture.

In some embodiments, an antibody provided herein may have one or morepost-translational modifications. In some embodiments, N-terminalcyclization, also called pyroglutamate formation (pyro-Glu), may occurin the antibody at N-terminal Glutamate (Glu) and/or Glutamine (Gln)residues during production. As such, it should be appreciated that anantibody specified as having a sequence comprising an N-terminalglutamate or glutamine residue encompasses antibodies that haveundergone pyroglutamate formation resulting from a post-translationalmodification. In some embodiments, pyroglutamate formation occurs in aheavy chain sequence. In some embodiments, pyroglutamate formationoccurs in a light chain sequence.

b. Other Muscle-Targeting Antibodies

In some embodiments, the muscle-targeting antibody is an antibody thatspecifically binds hemojuvelin, caveolin-3, Duchenne muscular dystrophypeptide, myosin IIb, or CD63. In some embodiments, the muscle-targetingantibody is an antibody that specifically binds a myogenic precursorprotein. Exemplary myogenic precursor proteins include, withoutlimitation, ABCG2, M-Cadherin/Cadherin-15, Caveolin-1, CD34, FoxK1,Integrin alpha 7, Integrin alpha 7 beta 1, MYF-5, MyoD, Myogenin,NCAM-1/CD56, Pax3, Pax7, and Pax9. In some embodiments, themuscle-targeting antibody is an antibody that specifically binds askeletal muscle protein. Exemplary skeletal muscle proteins include,without limitation, alpha-Sarcoglycan, beta-Sarcoglycan, CalpainInhibitors, Creatine Kinase MM/CKMM, eIF5A, Enolase 2/Neuron-specificEnolase, epsilon-Sarcoglycan, FABP3/H-FABP, GDF-8/Myostatin,GDF-11/GDF-8, Integrin alpha 7, Integrin alpha 7 beta 1, Integrin beta1/CD29, MCAM/CD146, MyoD, Myogenin, Myosin Light Chain KinaseInhibitors, NCAM-1/CD56, and Troponin I. In some embodiments, themuscle-targeting antibody is an antibody that specifically binds asmooth muscle protein. Exemplary smooth muscle proteins include, withoutlimitation, alpha-Smooth Muscle Actin, VE-Cadherin, Caldesmon/CALD1,Calponin 1, Desmin, Histamine H2 R, Motilin R/GPR38, Transgelin/TAGLN,and Vimentin. However, it should be appreciated that antibodies toadditional targets are within the scope of this disclosure and theexemplary lists of targets provided herein are not meant to be limiting.

c. Antibody Features/Alterations

In some embodiments, conservative mutations can be introduced intoantibody sequences (e.g., CDRs or framework sequences) at positionswhere the residues are not likely to be involved in interacting with atarget antigen (e.g., transferrin receptor), for example, as determinedbased on a crystal structure. In some embodiments, one, two or moremutations (e.g., amino acid substitutions) are introduced into the Fcregion of a muscle-targeting antibody described herein (e.g., in a CH2domain (residues 231-340 of human IgG1) and/or (e.g., and) CH3 domain(residues 341-447 of human IgG1) and/or (e.g., and) the hinge region,with numbering according to the Kabat numbering system (e.g., the EUindex in Kabat)) to alter one or more functional properties of theantibody, such as serum half-life, complement fixation, Fc receptorbinding and/or (e.g., and) antigen-dependent cellular cytotoxicity.

In some embodiments, one, two or more mutations (e.g., amino acidsubstitutions) are introduced into the hinge region of the Fc region(CH1 domain) such that the number of cysteine residues in the hingeregion are altered (e.g., increased or decreased) as described in, e.g.,U.S. Pat. No. 5,677,425. The number of cysteine residues in the hingeregion of the CH1 domain can be altered to, e.g., facilitate assembly ofthe light and heavy chains, or to alter (e.g., increase or decrease) thestability of the antibody or to facilitate linker conjugation.

In some embodiments, one, two or more mutations (e.g., amino acidsubstitutions) are introduced into the Fc region of a muscle-targetingantibody described herein (e.g., in a CH2 domain (residues 231-340 ofhuman IgG1) and/or (e.g., and) CH3 domain (residues 341-447 of humanIgG1) and/or (e.g., and) the hinge region, with numbering according tothe Kabat numbering system (e.g., the EU index in Kabat)) to increase ordecrease the affinity of the antibody for an Fc receptor (e.g., anactivated Fc receptor) on the surface of an effector cell. Mutations inthe Fc region of an antibody that decrease or increase the affinity ofan antibody for an Fc receptor and techniques for introducing suchmutations into the Fc receptor or fragment thereof are known to one ofskill in the art. Examples of mutations in the Fc receptor of anantibody that can be made to alter the affinity of the antibody for anFc receptor are described in, e.g., Smith P et al., (2012) PNAS 109:6181-6186, U.S. Pat. No. 6,737,056, and International Publication Nos.WO 02/060919; WO 98/23289; and WO 97/34631, which are incorporatedherein by reference.

In some embodiments, one, two or more amino acid mutations (i.e.,substitutions, insertions or deletions) are introduced into an IgGconstant domain, or FcRn-binding fragment thereof (preferably an Fc orhinge-Fc domain fragment) to alter (e.g., decrease or increase)half-life of the antibody in vivo. See, e.g., International PublicationNos. WO 02/060919; WO 98/23289; and WO 97/34631; and U.S. Pat. Nos.5,869,046, 6,121,022, 6,277,375 and 6,165,745 for examples of mutationsthat will alter (e.g., decrease or increase) the half-life of anantibody in vivo.

In some embodiments, one, two or more amino acid mutations (i.e.,substitutions, insertions or deletions) are introduced into an IgGconstant domain, or FcRn-binding fragment thereof (preferably an Fc orhinge-Fc domain fragment) to decrease the half-life of theanti-transferrin receptor antibody in vivo. In some embodiments, one,two or more amino acid mutations (i.e., substitutions, insertions ordeletions) are introduced into an IgG constant domain, or FcRn-bindingfragment thereof (preferably an Fc or hinge-Fc domain fragment) toincrease the half-life of the antibody in vivo. In some embodiments, theantibodies can have one or more amino acid mutations (e.g.,substitutions) in the second constant (CH2) domain (residues 231-340 ofhuman IgG1) and/or (e.g., and) the third constant (CH3) domain (residues341-447 of human IgG1), with numbering according to the EU index inKabat (Kabat E A et al., (1991) supra). In some embodiments, theconstant region of the IgG1 of an antibody described herein comprises amethionine (M) to tyrosine (Y) substitution in position 252, a serine(S) to threonine (T) substitution in position 254, and a threonine (T)to glutamic acid (E) substitution in position 256, numbered according tothe EU index as in Kabat. See U.S. Pat. No. 7,658,921, which isincorporated herein by reference. This type of mutant IgG, referred toas “YTE mutant” has been shown to display fourfold increased half-lifeas compared to wild-type versions of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281: 23514-24). In some embodiments, anantibody comprises an IgG constant domain comprising one, two, three ormore amino acid substitutions of amino acid residues at positions251-257, 285-290, 308-314, 385-389, and 428-436, numbered according tothe EU index as in Kabat.

In some embodiments, one, two or more amino acid substitutions areintroduced into an IgG constant domain Fc region to alter the effectorfunction(s) of the anti-transferrin receptor antibody. The effectorligand to which affinity is altered can be, for example, an Fc receptoror the Cl component of complement. This approach is described in furtherdetail in U.S. Pat. Nos. 5,624,821 and 5,648,260. In some embodiments,the deletion or inactivation (through point mutations or other means) ofa constant region domain can reduce Fc receptor binding of thecirculating antibody thereby increasing tumor localization. See, e.g.,U.S. Pat. Nos. 5,585,097 and 8,591,886 for a description of mutationsthat delete or inactivate the constant domain and thereby increase tumorlocalization. In some embodiments, one or more amino acid substitutionsmay be introduced into the Fc region of an antibody described herein toremove potential glycosylation sites on Fc region, which may reduce Fcreceptor binding (see, e.g., Shields R L et al., (2001) J Biol Chem 276:6591-604).

In some embodiments, one or more amino in the constant region of amuscle-targeting antibody described herein can be replaced with adifferent amino acid residue such that the antibody has altered C1qbinding and/or (e.g., and) reduced or abolished complement dependentcytotoxicity (CDC). This approach is described in further detail in U.S.Pat. No. 6,194,551 (Idusogie et al). In some embodiments, one or moreamino acid residues in the N-terminal region of the CH2 domain of anantibody described herein are altered to thereby alter the ability ofthe antibody to fix complement. This approach is described further inInternational Publication No. WO 94/29351. In some embodiments, the Fcregion of an antibody described herein is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or (e.g., and) to increase the affinity of theantibody for an Fcγ receptor. This approach is described further inInternational Publication No. WO 00/42072.

In some embodiments, the heavy and/or (e.g., and) light chain variabledomain(s) sequence(s) of the antibodies provided herein can be used togenerate, for example, CDR-grafted, chimeric, humanized, or compositehuman antibodies or antigen-binding fragments, as described elsewhereherein. As understood by one of ordinary skill in the art, any variant,CDR-grafted, chimeric, humanized, or composite antibodies derived fromany of the antibodies provided herein may be useful in the compositionsand methods described herein and will maintain the ability tospecifically bind transferrin receptor, such that the variant,CDR-grafted, chimeric, humanized, or composite antibody has at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95% or more binding to transferrin receptor relative to the originalantibody from which it is derived.

In some embodiments, the antibodies provided herein comprise mutationsthat confer desirable properties to the antibodies. For example, toavoid potential complications due to Fab-arm exchange, which is known tooccur with native IgG4 mAbs, the antibodies provided herein may comprisea stabilizing ‘Adair’ mutation (Angal S., et al., “A single amino acidsubstitution abolishes the heterogeneity of chimeric mouse/human (IgG4)antibody,” Mol Immunol 30, 105-108; 1993), where serine 228 (EUnumbering; residue 241 Kabat numbering) is converted to prolineresulting in an IgG1-like hinge sequence. Accordingly, any of theantibodies may include a stabilizing ‘Adair’ mutation.

As provided herein, antibodies of this disclosure may optionallycomprise constant regions or parts thereof. For example, a VL domain maybe attached at its C-terminal end to a light chain constant domain likeCκ or Cλ. Similarly, a VH domain or portion thereof may be attached toall or part of a heavy chain like IgA, IgD, IgE, IgG, and IgM, and anyisotype subclass. Antibodies may include suitable constant regions (see,for example, Kabat et al., Sequences of Proteins of ImmunologicalInterest, No. 91-3242, National Institutes of Health Publications,Bethesda, Md. (1991)). Therefore, antibodies within the scope of thismay disclosure include VH and VL domains, or an antigen binding portionthereof, combined with any suitable constant regions.

ii. Muscle-Targeting Peptides

Some aspects of the disclosure provide muscle-targeting peptides asmuscle-targeting agents. Short peptide sequences (e.g., peptidesequences of 5-20 amino acids in length) that bind to specific celltypes have been described. For example, cell-targeting peptides havebeen described in Vines e., et al., A. “Cell-penetrating andcell-targeting peptides in drug delivery” Biochim Biophys Acta 2008,1786: 126-38; Jarver P., et al., “In vivo biodistribution and efficacyof peptide mediated delivery” Trends Pharmacol Sci 2010; 31: 528-35;Samoylova T. I., et al., “Elucidation of muscle-binding peptides byphage display screening” Muscle Nerve 1999; 22: 460-6; U.S. Pat. No.6,329,501, issued on Dec. 11, 2001, entitled “METHODS AND COMPOSITIONSFOR TARGETING COMPOUNDS TO MUSCLE”; and Samoylov A. M., et al.,“Recognition of cell-specific binding of phage display derived peptidesusing an acoustic wave sensor.” Biomol Eng 2002; 18: 269-72; the entirecontents of each of which are incorporated herein by reference. Bydesigning peptides to interact with specific cell surface antigens(e.g., receptors), selectivity for a desired tissue, e.g., muscle, canbe achieved. Skeletal muscle-targeting has been investigated and a rangeof molecular payloads are able to be delivered. These approaches mayhave high selectivity for muscle tissue without many of the practicaldisadvantages of a large antibody or viral particle. Accordingly, insome embodiments, the muscle-targeting agent is a muscle-targetingpeptide that is from 4 to 50 amino acids in length. In some embodiments,the muscle-targeting peptide is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or50 amino acids in length. Muscle-targeting peptides can be generatedusing any of several methods, such as phage display.

In some embodiments, a muscle-targeting peptide may bind to aninternalizing cell surface receptor that is overexpressed or relativelyhighly expressed in muscle cells, e.g. a transferrin receptor, comparedwith certain other cells. In some embodiments, a muscle-targetingpeptide may target, e.g., bind to, a transferrin receptor. In someembodiments, a peptide that targets a transferrin receptor may comprisea segment of a naturally occurring ligand, e.g., transferrin. In someembodiments, a peptide that targets a transferrin receptor is asdescribed in U.S. Pat. No. 6,743,893, filed Nov. 30, 2000,“RECEPTOR-MEDIATED UPTAKE OF PEPTIDES THAT BIND THE HUMAN TRANSFERRINRECEPTOR”. In some embodiments, a peptide that targets a transferrinreceptor is as described in Kawamoto, M. et al, “A novel transferrinreceptor-targeted hybrid peptide disintegrates cancer cell membrane toinduce rapid killing of cancer cells.” BMC Cancer. 2011 Aug. 18; 11:359.In some embodiments, a peptide that targets a transferrin receptor is asdescribed in U.S. Pat. No. 8,399,653, filed May 20, 2011,“TRANSFERRIN/TRANSFERRIN RECEPTOR-MEDIATED SIRNA DELIVERY”.

As discussed above, examples of muscle targeting peptides have beenreported. For example, muscle-specific peptides were identified usingphage display library presenting surface heptapeptides. As one example apeptide having the amino acid sequence ASSLNIA (SEQ ID NO: 138) bound toC2C12 murine myotubes in vitro, and bound to mouse muscle tissue invivo. Accordingly, in some embodiments, the muscle-targeting agentcomprises the amino acid sequence ASSLNIA (SEQ ID NO: 138). This peptidedisplayed improved specificity for binding to heart and skeletal muscletissue after intravenous injection in mice with reduced binding toliver, kidney, and brain. Additional muscle-specific peptides have beenidentified using phage display. For example, a 12 amino acid peptide wasidentified by phage display library for muscle targeting in the contextof treatment for DMD. See, Yoshida D., et al., “Targeting of salicylateto skin and muscle following topical injections in rats.” Int J Pharm2002; 231: 177-84; the entire contents of which are hereby incorporatedby reference. Here, a 12 amino acid peptide having the sequenceSKTFNTHPQSTP (SEQ ID NO: 139) was identified and this muscle-targetingpeptide showed improved binding to C2C12 cells relative to the ASSLNIA(SEQ ID NO: 138) peptide.

An additional method for identifying peptides selective for muscle(e.g., skeletal muscle) over other cell types includes in vitroselection, which has been described in Ghosh D., et al., “Selection ofmuscle-binding peptides from context-specific peptide-presenting phagelibraries for adenoviral vector targeting” J Virol 2005; 79: 13667-72;the entire contents of which are incorporated herein by reference. Bypre-incubating a random 12-mer peptide phage display library with amixture of non-muscle cell types, non-specific cell binders wereselected out. Following rounds of selection the 12 amino acid peptideTARGEHKEEELI (SEQ ID NO: 140) appeared most frequently. Accordingly, insome embodiments, the muscle-targeting agent comprises the amino acidsequence TARGEHKEEELI (SEQ ID NO: 140).

A muscle-targeting agent may an amino acid-containing molecule orpeptide. A muscle-targeting peptide may correspond to a sequence of aprotein that preferentially binds to a protein receptor found in musclecells. In some embodiments, a muscle-targeting peptide contains a highpropensity of hydrophobic amino acids, e.g. valine, such that thepeptide preferentially targets muscle cells. In some embodiments, amuscle-targeting peptide has not been previously characterized ordisclosed. These peptides may be conceived of, produced, synthesized,and/or (e.g., and) derivatized using any of several methodologies, e.g.phage displayed peptide libraries, one-bead one-compound peptidelibraries, or positional scanning synthetic peptide combinatoriallibraries. Exemplary methodologies have been characterized in the artand are incorporated by reference (Gray, B. P. and Brown, K. C.“Combinatorial Peptide Libraries: Mining for Cell-Binding Peptides” ChemRev. 2014, 114:2, 1020-1081; Samoylova, T. I. and Smith, B. F.“Elucidation of muscle-binding peptides by phage display screening.”Muscle Nerve, 1999, 22:4. 460-6). In some embodiments, amuscle-targeting peptide has been previously disclosed (see, e.g. WriterM. J. et al. “Targeted gene delivery to human airway epithelial cellswith synthetic vectors incorporating novel targeting peptides selectedby phage display.” J. Drug Targeting. 2004; 12:185; Cal, D.“BDNF-mediated enhancement of inflammation and injury in the agingheart.” Physiol Genomics. 2006, 24:3, 191-7; Zhang, L. “Molecularprofiling of heart endothelial cells.” Circulation, 2005, 112:11,1601-11; McGuire, M. J. et al. “In vitro selection of a peptide withhigh selectivity for cardiomyocytes in vivo.” J Mol Biol. 2004, 342:1,171-82). Exemplary muscle-targeting peptides comprise an amino acidsequence of the following group: CQAQGQLVC (SEQ ID NO: 141), CSERSMNFC(SEQ ID NO: 142), CPKTRRVPC (SEQ ID NO: 143), WLSEAGPVVTVRALRGTGSW (SEQID NO: 144), ASSLNIA (SEQ ID NO: 138), CMQHSMRVC (SEQ ID NO: 145), andDDTRHWG (SEQ ID NO: 146). In some embodiments, a muscle-targetingpeptide may comprise about 2-25 amino acids, about 2-20 amino acids,about 2-15 amino acids, about 2-10 amino acids, or about 2-5 aminoacids. Muscle-targeting peptides may comprise naturally-occurring aminoacids, e.g. cysteine, alanine, or non-naturally-occurring or modifiedamino acids. Non-naturally occurring amino acids include β-amino acids,homo-amino acids, proline derivatives, 3-substituted alaninederivatives, linear core amino acids, N-methyl amino acids, and othersknown in the art. In some embodiments, a muscle-targeting peptide may belinear; in other embodiments, a muscle-targeting peptide may be cyclic,e.g. bicyclic (see, e.g. Silvana, M. G. et al. Mol. Therapy, 2018, 26:1,132-147).

iii. Muscle-Targeting Receptor Ligands

A muscle-targeting agent may be a ligand, e.g. a ligand that binds to areceptor protein. A muscle-targeting ligand may be a protein, e.g.transferrin, which binds to an internalizing cell surface receptorexpressed by a muscle cell. Accordingly, in some embodiments, themuscle-targeting agent is transferrin, or a derivative thereof thatbinds to a transferrin receptor. A muscle-targeting ligand mayalternatively be a small molecule, e.g. a lipophilic small molecule thatpreferentially targets muscle cells relative to other cell types.Exemplary lipophilic small molecules that may target muscle cellsinclude compounds comprising cholesterol, cholesteryl, stearic acid,palmitic acid, oleic acid, oleyl, linolene, linoleic acid, myristicacid, sterols, dihydrotestosterone, testosterone derivatives, glycerine,alkyl chains, trityl groups, and alkoxy acids.

iv. Muscle-Targeting Aptamers

A muscle-targeting agent may be an aptamer, e.g. an RNA aptamer, whichpreferentially targets muscle cells relative to other cell types. Insome embodiments, a muscle-targeting aptamer has not been previouslycharacterized or disclosed. These aptamers may be conceived of,produced, synthesized, and/or (e.g., and) derivatized using any ofseveral methodologies, e.g. Systematic Evolution of Ligands byExponential Enrichment. Exemplary methodologies have been characterizedin the art and are incorporated by reference (Yan, A. C. and Levy, M.“Aptamers and aptamer targeted delivery” RNA biology, 2009, 6:3, 316-20;Germer, K. et al. “RNA aptamers and their therapeutic and diagnosticapplications.” Int. J. Biochem. Mol. Biol. 2013; 4: 27-40). In someembodiments, a muscle-targeting aptamer has been previously disclosed(see, e.g. Phillippou, S. et al. “Selection and Identification ofSkeletal-Muscle-Targeted RNA Aptamers.” Mol Ther Nucleic Acids. 2018,10:199-214; Thiel, W. H. et al. “Smooth Muscle Cell-targeted RNA AptamerInhibits Neointimal Formation.” Mol Ther. 2016, 24:4, 779-87). Exemplarymuscle-targeting aptamers include the A01B RNA aptamer and RNA Apt 14.In some embodiments, an aptamer is a nucleic acid-based aptamer, anoligonucleotide aptamer or a peptide aptamer. In some embodiments, anaptamer may be about 5-15 kDa, about 5-10 kDa, about 10-15 kDa, about1-5 Da, about 1-3 kDa, or smaller.

v. Other Muscle-Targeting Agents

One strategy for targeting a muscle cell (e.g., a skeletal muscle cell)is to use a substrate of a muscle transporter protein, such as atransporter protein expressed on the sarcolemma. In some embodiments,the muscle-targeting agent is a substrate of an influx transporter thatis specific to muscle tissue. In some embodiments, the influxtransporter is specific to skeletal muscle tissue. Two main classes oftransporters are expressed on the skeletal muscle sarcolemma, (1) theadenosine triphosphate (ATP) binding cassette (ABC) superfamily, whichfacilitate efflux from skeletal muscle tissue and (2) the solute carrier(SLC) superfamily, which can facilitate the influx of substrates intoskeletal muscle. In some embodiments, the muscle-targeting agent is asubstrate that binds to an ABC superfamily or an SLC superfamily oftransporters. In some embodiments, the substrate that binds to the ABCor SLC superfamily of transporters is a naturally-occurring substrate.In some embodiments, the substrate that binds to the ABC or SLCsuperfamily of transporters is a non-naturally occurring substrate, forexample, a synthetic derivative thereof that binds to the ABC or SLCsuperfamily of transporters.

In some embodiments, the muscle-targeting agent is a substrate of an SLCsuperfamily of transporters. SLC transporters are either equilibrativeor use proton or sodium ion gradients created across the membrane todrive transport of substrates. Exemplary SLC transporters that have highskeletal muscle expression include, without limitation, the SATTtransporter (ASCT1; SLC1A4), GLUT4 transporter (SLC2A4), GLUT7transporter (GLUT7; SLC2A7), ATRC2 transporter (CAT-2; SLC7A2), LAT3transporter (KIAA0245; SLC7A6), PHT1 transporter (PTR4; SLC15A4), OATP-Jtransporter (OATP5A1; SLC21A15), OCT3 transporter (EMT; SLC22A3), OCTN2transporter (FLJ46769; SLC22A5), ENT transporters (ENT1; SLC29A1 andENT2; SLC29A2), PAT2 transporter (SLC36A2), and SAT2 transporter(KIAA1382; SLC38A2). These transporters can facilitate the influx ofsubstrates into skeletal muscle, providing opportunities for muscletargeting.

In some embodiments, the muscle-targeting agent is a substrate of anequilibrative nucleoside transporter 2 (ENT2) transporter. Relative toother transporters, ENT2 has one of the highest mRNA expressions inskeletal muscle. While human ENT2 (hENT2) is expressed in most bodyorgans such as brain, heart, placenta, thymus, pancreas, prostate, andkidney, it is especially abundant in skeletal muscle. Human ENT2facilitates the uptake of its substrates depending on theirconcentration gradient. ENT2 plays a role in maintaining nucleosidehomeostasis by transporting a wide range of purine and pyrimidinenucleobases. The hENT2 transporter has a low affinity for allnucleosides (adenosine, guanosine, uridine, thymidine, and cytidine)except for inosine. Accordingly, in some embodiments, themuscle-targeting agent is an ENT2 substrate. Exemplary ENT2 substratesinclude, without limitation, inosine, 2′,3′-dideoxyinosine, andcalofarabine. In some embodiments, any of the muscle-targeting agentsprovided herein are associated with a molecular payload (e.g.,oligonucleotide payload). In some embodiments, the muscle-targetingagent is covalently linked to the molecular payload. In someembodiments, the muscle-targeting agent is non-covalently linked to themolecular payload.

In some embodiments, the muscle-targeting agent is a substrate of anorganic cation/carnitine transporter (OCTN2), which is a sodiumion-dependent, high affinity carnitine transporter. In some embodiments,the muscle-targeting agent is carnitine, mildronate, acetylcarnitine, orany derivative thereof that binds to OCTN2. In some embodiments, thecarnitine, mildronate, acetylcarnitine, or derivative thereof iscovalently linked to the molecular payload (e.g., oligonucleotidepayload).

A muscle-targeting agent may be a protein that is protein that exists inat least one soluble form that targets muscle cells. In someembodiments, a muscle-targeting protein may be hemojuvelin (also knownas repulsive guidance molecule C or hemochromatosis type 2 protein), aprotein involved in iron overload and homeostasis. In some embodiments,hemojuvelin may be full length or a fragment, or a mutant with at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98% or at least 99% sequence identity to a functional hemojuvelinprotein. In some embodiments, a hemojuvelin mutant may be a solublefragment, may lack a N-terminal signaling, and/or (e.g., and) lack aC-terminal anchoring domain. In some embodiments, hemojuvelin may beannotated under GenBank RefSeq Accession Numbers NM_001316767.1,NM_145277.4, NM_202004.3, NM_213652.3, or NM_213653.3. It should beappreciated that a hemojuvelin may be of human, non-human primate, orrodent origin.

B. Molecular Payloads

Some aspects of the disclosure provide molecular payloads, e.g., formodulating a biological outcome, e.g., the transcription of a DNAsequence, the expression of a protein, or the activity of a protein. Insome embodiments, a molecular payload is linked to, or otherwiseassociated with a muscle-targeting agent. In some embodiments, suchmolecular payloads are capable of targeting to a muscle cell, e.g., viaspecifically binding to a nucleic acid or protein in the muscle cellfollowing delivery to the muscle cell by an associated muscle-targetingagent. It should be appreciated that various types of muscle-targetingagents may be used in accordance with the disclosure. For example, themolecular payload may comprise, or consist of, an oligonucleotide (e.g.,antisense oligonucleotide), a peptide (e.g., a peptide that binds anucleic acid or protein associated with disease in a muscle cell), aprotein (e.g., a protein that binds a nucleic acid or protein associatedwith disease in a muscle cell), or a small molecule (e.g., a smallmolecule that modulates the function of a nucleic acid or proteinassociated with disease in a muscle cell). In some embodiments, themolecular payload is an oligonucleotide that comprises a strand having aregion of complementarity to a DMPK allele comprising adisease-associated-repeat expansion. Exemplary molecular payloads aredescribed in further detail herein, however, it should be appreciatedthat the exemplary molecular payloads provided herein are not meant tobe limiting.

i. Oligonucleotides

Any suitable oligonucleotide may be used as a molecular payload, asdescribed herein. In some embodiments, the oligonucleotide may bedesigned to cause degradation of an mRNA (e.g., the oligonucleotide maybe a gapmer, an siRNA, a ribozyme or an aptamer that causesdegradation). In some embodiments, the oligonucleotide may be designedto block translation of an mRNA (e.g., the oligonucleotide may be amixmer, an siRNA or an aptamer that blocks translation). In someembodiments, an oligonucleotide may be designed to caused degradationand block translation of an mRNA. In some embodiments, anoligonucleotide may be a guide nucleic acid (e.g., guide RNA) fordirecting activity of an enzyme (e.g., a gene editing enzyme). Otherexamples of oligonucleotides are provided herein. It should beappreciated that, in some embodiments, oligonucleotides in one format(e.g., antisense oligonucleotides) may be suitably adapted to anotherformat (e.g., siRNA oligonucleotides) by incorporating functionalsequences (e.g., antisense strand sequences) from one format to theother format.

Examples of oligonucleotides useful for targeting DMPK are provided inUS Patent Application Publication 20100016215A1, published on Jan. 1,2010, entitled Compound And Method For Treating Myotonic Dystrophy; USPatent Application Publication 20130237585A1, published Jul. 19, 2010,Modulation Of Dystrophia Myotonica-Protein Kinase (DMPK) Expression; USPatent Application Publication 20150064181A1, published on Mar. 5, 2015,entitled “Antisense Conjugates For Decreasing Expression Of Dmpk”; USPatent Application Publication 20150238627A1, published on Aug. 27,2015, entitled “Peptide-Linked Morpholino Antisense Oligonucleotides ForTreatment Of Myotonic Dystrophy”; and US Patent Application Publication20160304877A1, published on Oct. 20, 2016, entitled “Compounds AndMethods For Modulation Of Dystrophia Myotonica-Protein Kinase (Dmpk)Expression,” the contents of each of which are incorporated herein intheir entireties.

Examples of oligonucleotides for promoting DMPK gene editing include USPatent Application Publication 20170088819A1, published on Mar. 3, 2017,entitled “Genetic Correction Of Myotonic Dystrophy Type 1”; andInternational Patent Application Publication WO18002812A1, published onApr. 1, 2018, entitled “Materials And Methods For Treatment Of MyotonicDystrophy Type 1 (DM1) And Other Related Disorders,” the contents ofeach of which are incorporated herein in their entireties.

In some embodiments, oligonucleotides may have a region ofcomplementarity to a sequence set forth as follows, which is an examplehuman DMPK gene sequence (Gene ID 1760: NM_001081560.21:

(SEQ ID NO: 131) AGGGGGGCTGGACCAAGGGGTGGGGAGAAGGGGAGGAGGCCTCGGCCGGCCGCAGAGAGAAGTGGCCAGAGAGGCCCAGGGGACAGCCAGGGACAGGCAGACATGCAGCCAGGGCTCCAGGGCCTGGACAGGGGCTGCCAGGCCCTGTGACAGGAGGACCCCGAGCCCCCGGCCCGGGGAGGGGCCATGGTGCTGCCTGTCCAACATGTCAGCCGAGGTGCGGCTGAGGCGGCTCCAGCAGCTGGTGTTGGACCCGGGCTTCCTGGGGCTGGAGCCCCTGCTCGACCTTCTCCTGGGCGTCCACCAGGAGCTGGGCGCCTCCGAACTGGCCCAGGACAAGTACGTGGCCGACTTCTTGCAGTGGGCGGAGCCCATCGTGGTGAGGCTTAAGGAGGTCCGACTGCAGAGGGACGACTTCGAGATTCTGAAGGTGATCGGACGCGGGGCGTTCAGCGAGGTAGCGGTAGTGAAGATGAAGCAGACGGGCCAGGTGTATGCCATGAAGATCATGAACAAGTGGGACATGCTGAAGAGGGGCGAGGTGTCGTGCTTCCGTGAGGAGAGGGACGTGTTGGTGAATGGGGACCGGCGGTGGATCACGCAGCTGCACTTCGCCTTCCAGGATGAGAACTACCTGTACCTGGTCATGGAGTATTACGTGGGCGGGGACCTGCTGACACTGCTGAGCAAGTTTGGGGAGCGGATTCCGGCCGAGATGGCGCGCTTCTACCTGGCGGAGATTGTCATGGCCATAGACTCGGTGCACCGGCTTGGCTACGTGCACAGGGACATCAAACCCGACAACATCCTGCTGGACCGCTGTGGCCACATCCGCCTGGCCGACTTCGGCTCTTGCCTCAAGCTGCGGGCAGATGGAACGGTGCGGTCGCTGGTGGCTGTGGGCACCCCAGACTACCTGTCCCCCGAGATCCTGCAGGCTGTGGGCGGTGGGCCTGGGACAGGCAGCTACGGGCCCGAGTGTGACTGGTGGGCGCTGGGTGTATTCGCCTATGAAATGTTCTATGGGCAGACGCCCTTCTACGCGGATTCCACGGCGGAGACCTATGGCAAGATCGTCCACTACAAGGAGCACCTCTCTCTGCCGCTGGTGGACGAAGGGGTCCCTGAGGAGGCTCGAGACTTCATTCAGCGGTTGCTGTGTCCCCCGGAGACACGGCTGGGCCGGGGTGGAGCAGGCGACTTCCGGACACATCCCTTCTTCTTTGGCCTCGACTGGGATGGTCTCCGGGACAGCGTGCCCCCCTTTACACCGGATTTCGAAGGTGCCACCGACACATGCAACTTCGACTTGGTGGAGGACGGGCTCACTGCCATGGAGACACTGTCGGACATTCGGGAAGGTGCGCCGCTAGGGGTCCACCTGCCTTTTGTGGGCTACTCCTACTCCTGCATGGCCCTCAGGGACAGTGAGGTCCCAGGCCCCACACCCATGGAACTGGAGGCCGAGCAGCTGCTTGAGCCACACGTGCAAGCGCCCAGCCTGGAGCCCTCGGTGTCCCCACAGGATGAAACAGCTGAAGTGGCAGTTCCAGCGGCTGTCCCTGCGGCAGAGGCTGAGGCCGAGGTGACGCTGCGGGAGCTCCAGGAAGCCCTGGAGGAGGAGGTGCTCACCCGGCAGAGCCTGAGCCGGGAGATGGAGGCCATCCGCACGGACAACCAGAACTTCGCCAGTCAACTACGCGAGGCAGAGGCTCGGAACCGGGACCTAGAGGCACACGTCCGGCAGTTGCAGGAGCGGATGGAGTTGCTGCAGGCAGAGGGAGCCACAGCTGTCACGGGGGTCCCCAGTCCCCGGGCCACGGATCCACCTTCCCATCTAGATGGCCCCCCGGCCGTGGCTGTGGGCCAGTGCCCGCTGGTGGGGCCAGGCCCCATGCACCGCCGCCACCTGCTGCTCCCTGCCAGGGTCCCTAGGCCTGGCCTATCGGAGGCGCTTTCCCTGCTCCTGTTCGCCGTTGTTCTGTCTCGTGCCGCCGCCCTGGGCTGCATTGGGTTGGTGGCCCACGCCGGCCAACTCACCGCAGTCTGGCGCCGCCCAGGAGCCGCCCGCGCTCCCTGAACCCTAGAACTGTCTTCGACTCCGGGGCCCCGTTGGAAGACTGAGTGCCCGGGGCACGGCACAGAAGCCGCGCCCACCGCCTGCCAGTTCACAACCGCTCCGAGCGTGGGTCTCCGCCCAGCTCCAGTCCTGTGATCCGGGCCCGCCCCCTAGCGGCCGGGGAGGGAGGGGCCGGGTCCGCGGCCGGCGAACGGGGCTCGAAGGGTCCTTGTAGCCGGGAATGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGGGGGGATCACAGACCATTTCTTTCTTTCGGCCAGGCTGAGGCCCTGACGTGGATGGGCAAACTGCAGGCCTGGGAAGGCAGCAAGCCGGGCCGTCCGTGTTCCATCCTCCACGCACCCCCACCTATCGTTGGTTCGCAAAGTGCAAAGCTTTCTTGTGCATGACGCCCTGCTCTGGGGAGCGTCTGGCGCGATCTCTGCCTGCTTACTCGGGAAATTTGCTTTTGCCAAACCCGCTTTTTCGGGGATCCCGCGCCCCCCTCCTCACTTGCGCTGCTCTCGGAGCCCCAGCCGGCTCCGCCCGCTTCGGCGGTTTGGATATTTATTGACCTCGTCCTCCGACTCGCTGACAGGCTACAGGACCCCCAACAACCCCAATCCACGTTTTGGATGCACTGAGACCCCGACATTCCTCGGTATTTATTGTCTGTCCCCACCTAGGACCCCCACCCCCGACCCTCGCGAATAAAAGGCCCTCCATCTGCCCAAAGCTCTGGA.

In some embodiments, oligonucleotides may have a region ofcomplementarity to a sequence set forth as follows, which is an examplemouse DMPK gene sequence (Gene ID 13400; NM_001190490.1).

(SEQ ID NO: 147) GAACTGGCCAGAGAGACCCAAGGGATAGTCAGGGACGGGCAGACATGCAGCTAGGGTTCTGGGGCCTGGACAGGGGCAGCCAGGCCCTGTGACGGGAAGACCCCGAGCTCCGGCCCGGGGAGGGGCCATGGTGTTGCCTGCCCAACATGTCAGCCGAAGTGCGGCTGAGGCAGCTCCAGCAGCTGGTGCTGGACCCAGGCTTCCTGGGACTGGAGCCCCTGCTCGACCTTCTCCTGGGCGTCCACCAGGAGCTGGGTGCCTCTCACCTAGCCCAGGACAAGTATGTGGCCGACTTCTTGCAGTGGGTGGAGCCCATTGCAGCAAGGCTTAAGGAGGTCCGACTGCAGAGGGATGATTTTGAGATTTTGAAGGTGATCGGGCGTGGGGCGTTCAGCGAGGTAGCGGTGGTGAAGATGAAACAGACGGGCCAAGTGTATGCCATGAAGATTATGAATAAGTGGGACATGCTGAAGAGAGGCGAGGTGTCGTGCTTCCGGGAAGAAAGGGATGTATTAGTGAAAGGGGACCGGCGCTGGATCACACAGCTGCACTTTGCCTTCCAGGATGAGAACTACCTGTACCTGGTCATGGAATACTACGTGGGCGGGGACCTGCTAACGCTGCTGAGCAAGTTTGGGGAGCGGATCCCCGCCGAGATGGCTCGCTTCTACCTGGCCGAGATTGTCATGGCCATAGACTCCGTGCACCGGCTGGGCTACGTGCACAGGGACATCAAACCAGATAACATTCTGCTGGACCGATGTGGGCACATTCGCCTGGCAGACTTCGGCTCCTGCCTCAAACTGCAGCCTGATGGAATGGTGAGGTCGCTGGTGGCTGTGGGCACCCCGGACTACCTGTCTCCTGAGATTCTGCAGGCCGTTGGTGGAGGGCCTGGGGCAGGCAGCTACGGGCCAGAGTGTGACTGGTGGGCACTGGGCGTGTTCGCCTATGAGATGTTCTATGGGCAGACCCCCTTCTACGCGGACTCCACAGCCGAGACATATGCCAAGATTGTGCACTACAGGGAACACTTGTCGCTGCCGCTGGCAGACACAGTTGTCCCCGAGGAAGCTCAGGACCTCATTCGTGGGCTGCTGTGTCCTGCTGAGATAAGGCTAGGTCGAGGTGGGGCAGACTTCGAGGGTGCCACGGACACATGCAATTTCGATGTGGTGGAGGACCGGCTCACTGCCATGGTGAGCGGGGGCGGGGAGACGCTGTCAGACATGCAGGAAGACATGCCCCTTGGGGTGCGCCTGCCCTTCGTGGGCTACTCCTACTGCTGCATGGCCTTCAGAGACAATCAGGTCCCGGACCCCACCCCTATGGAACTAGAGGCCCTGCAGTTGCCTGTGTCAGACTTGCAAGGGCTTGACTTGCAGCCCCCAGTGTCCCCACCGGATCAAGTGGCTGAAGAGGCTGACCTAGTGGCTGTCCCTGCCCCTGTGGCTGAGGCAGAGACCACGGTAACGCTGCAGCAGCTCCAGGAAGCCCTGGAAGAAGAGGTTCTCACCCGGCAGAGCCTGAGCCGCGAGCTGGAGGCCATCCGGACCGCCAACCAGAACTTCTCCAGCCAACTACAGGAGGCCGAGGTCCGAAACCGAGACCTGGAGGCGCATGTTCGGCAGCTACAGGAACGGATGGAGATGCTGCAGGCCCCAGGAGCCGCAGCCATCACGGGGGTCCCCAGTCCCCGGGCCACGGATCCACCTTCCCATCTAGATGGCCCCCCGGCCGTGGCTGTGGGCCAGTGCCCGCTGGTGGGGCCAGGCCCCATGCACCGCCGTCACCTGCTGCTCCCTGCCAGGATCCCTAGGCCTGGCCTATCCGAGGCGCGTTGCCTGCTCCTGTTCGCCGCTGCTCTGGCTGCTGCCGCCACACTGGGCTGCACTGGGTTGGTGGCCTATACCGGCGGTCTCACCCCAGTCTGGTGTTTCCCGGGAGCCACCTTCGCCCCCTGAACCCTAAGACTCCAAGCCATCTTTCATTTAGGCCTCCTAGGAAGGTCGAGCGACCAGGGAGCGACCCAAAGCGTCTCTGTGCCCATCGCGCCCCCCCCCCCCCCCCACCGCTCCGCTCCACACTTCTGTGAGCCTGGGTCCCCACCCAGCTCCGCTCCTGTGATCCAGGCCTGCCACCTGGCGGCCGGGGAGGGAGGAACAGGGCTCGTGCCCAGCACCCCTGGTTCCTGCAGAGCTGGTAGCCACCGCTGCTGCAGCAGCTGGGCATTCGCCGACCTTGCTTTACTCAGCCCCGACGTGGATGGGCAAACTGCTCAGCTCATCCGATTTCACTTTTTCACTCTCCCAGCCATCAGTTACAAGCCATAAGCATGAGCCCCCTATTTCCAGGGACATCCCATTCCCATAGTGATGGATCAGCAAGACCTCTGCCAGCACACACGGAGTCTTTGGCTTCGGACAGCCTCACTCCTGGGGGTTGCTGCAACTCCTTCCCCGTGTACACGTCTGCACTCTAACAACGGAGCCACAGCTGCACTCCCCCCTCCCCCAAAGCAGTGTGGGTATTTATTGATCTTGTTATCTGACTCACTGACAGACTCCGGGACCCACGTTTTAGATGCATTGAGACTCGACATTCCTCGGTATTTATTGTCTGTCCCCACCTACGACCTCCACTCCCGACCCTTGCGAATAAAATACTTCTGGTCTGCCCTAAAIn some embodiments, an oligonucleotide may have a region ofcomplementarity to DMPK gene sequences of multiple species, e.g.,selected from human, mouse and non-human species.

In some embodiments, the oligonucleotide may have region ofcomplementarity to a mutant form of DMPK, for example, a mutant form asreported in Botta A. et al. “The CTG repeat expansion size correlateswith the splicing defects observed in muscles from myotonic dystrophytype 1 patients.” J Med Genet. 2008 October; 45(10):639-46; andMachuca-Tzili L. et al. “Clinical and molecular aspects of the myotonicdystrophies: a review.” Muscle Nerve. 2005 July; 32(1):1-18; thecontents of each of which are incorporated herein by reference in theirentireties.

In some embodiments, the oligonucleotide may target lncRNA or mRNA,e.g., for degradation. In some embodiments, the oligonucleotide maytarget, e.g., for degradation, a nucleic acid encoding a proteininvolved in a mismatch repair pathway, e.g., MSH2, MutLalpha, MutSbeta,MutLalpha. Non-limiting examples of proteins involved in mismatch repairpathways, for which mRNAs encoding such proteins may be targeted byoligonucleotides described herein, are described in Iyer, R. R. et al.,“DNA triplet repeat expansion and mismatch repair” Annu Rev Biochem.2015; 84:199-226; and Schmidt M. H. and Pearson C. E.,“Disease-associated repeat instability and mismatch repair” DNA Repair(Amst). 2016 February; 38:117-26.

In some embodiments, an oligonucleotide provided herein is an antisenseoligonucleotide targeting DMPK. In some embodiments, the oligonucleotidetargeting is any one of the antisense oligonucleotides (e.g., a Gapmer)targeting DMPK as described in US Patent Application PublicationUS20160304877A1, published on Oct. 20, 2016, entitled “Compounds AndMethods For Modulation Of Dystrophia Myotonica-Protein Kinase (DMPK)Expression,” incorporated herein by reference). In some embodiments, theDMPK targeting oligonucleotide targets a region of the DMPK genesequence as set forth in Genbank accession No. NM_001081560.2 (SEQ IDNO: 131) or as set forth in Genbank accession No. NG_009784.1.

In some embodiments, the DMPK targeting oligonucleotide comprises anucleotide sequence comprising a region complementary to a target regionthat is at least 10 continuous nucleotides (e.g., at least 10, at least12, at least 14, at least 16, or more continuous nucleotides) in SEQ IDNO: 131.

In some embodiments, the DMPK targeting oligonucleotide comprise agapmer motif. “Gapmer” means a chimeric antisense compound in which aninternal region having a plurality of nucleotides that support RNase Hcleavage is positioned between external regions having one or morenucleotides, wherein the nucleotides comprising the internal region arechemically distinct from the nucleotide or nucleotides comprising theexternal regions. The internal region can be referred to as a “gapsegment” and the external regions can be referred to as “wing segments.”In some embodiments, the DMPK targeting oligonucleotide comprises one ormore modified nucleotides, and/or (e.g., and) one or more modifiedinternucleotide linkages. In some embodiments, the internucleotidelinkage is a phosphorothioate linkage. In some embodiments, theoligonucleotide comprises a full phosphorothioate backbone. In someembodiments, the oligonucleotide is a DNA gapmer with cET ends (e.g.,3-10-3; cET-DNA-cET). In some embodiments, the DMPK targetingoligonucleotide comprises one or more 6′-(S)—CH₃ biocyclic nucleotides,one or more β-D-2′-deoxyribonucleotides, and/or (e.g., and) one or more5-methylcytosine nucleotides.

In some embodiments, the DMPK targeting oligonucleotide is a gapmerhaving the formula 5′-X—Y—Z-3′, with X and Z as wing segments and Y asthe gap segment. In some embodiments, the DMPK targeting oligonucleotideis a gapmer having a 5′-4-8-4-3′ formula. In some embodiments, the DMPKtargeting oligonucleotide is a gapmer having a 5′-5-10-5-3′ formula. Insome embodiments, the DMPK targeting oligonucleotide is a gapmer havinga 5′-3-10-3-3′ formula. In some embodiments, the DMPK targetingoligonucleotide is a gapmer comprising one or more of 5-methylcytosinenucleotides, 2′OMe nucleotides, 2′fluoro nucleotides, LNAs, and/or(e.g., and) 2′-O-methoxyethyl (2′-O-MOE) nucleotides. In someembodiments, the DMPK targeting oligonucleotide is a gapmer comprisingone or more modified internucleotide (e.g., a phosphorothioate linkage).In some embodiments, the DMPK targeting oligonucleotide is a gapmercomprising a full phosphorothioate backbone.

In some embodiments, any one of the oligonucleotides can be in saltform, e.g., as sodium, potassium, or magnesium salts.

In some embodiments, the 5′ or 3′ nucleoside (e.g., terminal nucleoside)of any one of the oligonucleotides described herein is conjugated to anamine group, optionally via a spacer. In some embodiments, the spacercomprises an aliphatic moiety. In some embodiments, the spacer comprisesa polyethylene glycol moiety. In some embodiments, a phosphodiesterlinkage is present between the spacer and the 5′ or 3′ nucleoside of theoligonucleotide. In some embodiments, the 5′ or 3′ nucleoside (e.g.,terminal nucleoside) of any of the oligonucleotides described herein isconjugated to a spacer that is a substituted or unsubstituted aliphatic,substituted or unsubstituted heteroaliphatic, substituted orunsubstituted carbocyclylene, substituted or unsubstitutedheterocyclylene, substituted or unsubstituted arylene, substituted orunsubstituted heteroarylene, —O—, —N(R^(A))—, —S—, —C(═O)—, —C(═O)O—,—C(═O)NR^(A)—, —NR^(A)C(═O)—, —NR^(A)C(═O)R^(A)—, —C(═O)R^(A)—,—NR^(A)C(═O)O—, —NR^(A)C(═O)N(R^(A))—, —OC(═O)—, —OC(═O)O—,—OC(═O)N(R^(A))—, —S(O)₂NR^(A)—, —NR^(A)S(O)₂—, or a combinationthereof; each R^(A) is independently hydrogen or substituted orunsubstituted alkyl. In certain embodiments, the spacer is a substitutedor unsubstituted alkylene, substituted or unsubstituted heterocyclylene,substituted or unsubstituted heteroarylene, —O—, —N(R^(A))—, or—C(═O)N(R^(A))₂, or a combination thereof.

In some embodiments, the 5′ or 3′ nucleoside of any one of theoligonucleotides described herein is conjugated to a compound of theformula —NH₂—(CH₂)_(n)—, wherein n is an integer from 1 to 12. In someembodiments, n is 6, 7, 8, 9, 10, 11, or 12. In some embodiments, aphosphodiester linkage is present between the compound of the formulaNH₂—(CH₂)_(n)— and the 5′ or 3′ nucleoside of the oligonucleotide. Insome embodiments, a compound of the formula NH₂—(CH₂)₆— is conjugated tothe oligonucleotide via a reaction between 6-amino-1-hexanol(NH₂—(CH₂)₆—OH) and the 5′ phosphate of the oligonucleotide.

In some embodiments, the oligonucleotide is conjugated to a targetingagent, e.g., a muscle targeting agent such as an anti-TfR antibody,e.g., via the amine group.

a. Oligonucleotide Size/Sequence

Oligonucleotides may be of a variety of different lengths, e.g.,depending on the format. In some embodiments, an oligonucleotide is 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length.In some embodiments, the oligonucleotide is 8 to 50 nucleotides inlength, 8 to 40 nucleotides in length, 8 to 30 nucleotides in length, 10to 15 nucleotides in length, 10 to 20 nucleotides in length, 15 to 25nucleotides in length, 21 to 23 nucleotides in lengths, etc.

In some embodiments, a complementary nucleic acid sequence of anoligonucleotide for purposes of the present disclosure is specificallyhybridizable or specific for the target nucleic acid when binding of thesequence to the target molecule (e.g., mRNA) interferes with the normalfunction of the target (e.g., mRNA) to cause a loss of activity (e.g.,inhibiting translation) or expression (e.g., degrading a target mRNA)and there is a sufficient degree of complementarity to avoidnon-specific binding of the sequence to non-target sequences underconditions in which avoidance of non-specific binding is desired, e.g.,under physiological conditions in the case of in vivo assays ortherapeutic treatment, and in the case of in vitro assays, underconditions in which the assays are performed under suitable conditionsof stringency. Thus, in some embodiments, an oligonucleotide may be atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% complementary to the consecutivenucleotides of a target nucleic acid. In some embodiments acomplementary nucleotide sequence need not be 100% complementary to thatof its target to be specifically hybridizable or specific for a targetnucleic acid.

In some embodiments, an oligonucleotide comprises region ofcomplementarity to a target nucleic acid that is in the range of 8 to15, 8 to 30, 8 to 40, or 10 to 50, or 5 to 50, or 5 to 40 nucleotides inlength. In some embodiments, a region of complementarity of anoligonucleotide to a target nucleic acid is 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, or 50 nucleotides in length. In some embodiments, the region ofcomplementarity is complementary with at least 8 consecutive nucleotidesof a target nucleic acid. In some embodiments, an oligonucleotide maycontain 1, 2 or 3 base mismatches compared to the portion of theconsecutive nucleotides of target nucleic acid. In some embodiments theoligonucleotide may have up to 3 mismatches over 15 bases, or up to 2mismatches over 10 bases.

In some embodiments, an oligonucleotide comprises at least 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides of a sequencecomprising any one of SEQ ID NO: 148-383 and 621-638. In someembodiments, an oligonucleotide comprises a sequence comprising any oneof SEQ ID NO: 148-383 and 621-638. In some embodiments, anoligonucleotide comprises a sequence that shares at least 70%, 75%, 80%,85%, 90%, 95%, or 97% sequence identity with at least 12 or at least 15consecutive nucleotides of any one of SEQ ID NO: 148-383 and 621-638.

In some embodiments, an oligonucleotide comprises a sequence thattargets a DMPK sequence comprising any one of SEQ ID NO: 384-619. Insome embodiments, an oligonucleotide comprises at least 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 nucleotides (e.g., consecutivenucleotides) that are complementary to a DMPK sequence comprising anyone of SEQ ID NO: 384-619. In some embodiments, an oligonucleotidecomprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or97% complementary with at least 12 or at least 15 consecutivenucleotides of any one of SEQ ID NO: 384-619.

In some embodiments, the oligonucleotide is complementary (e.g., atleast 85% at least 90%, at least 95%, or 100%) to a target sequence ofany one of the oligonucleotides provided herein (e.g., theoligonucleotides listed in Table 8 or Table 17). In some embodiments,such target sequence is 100% complementary to the oligonucleotide listedin Table 8 or Table 17.

In some embodiments, any one or more of the thymine bases (T's) in anyone of the oligonucleotides provided herein (e.g., the oligonucleotideslisted in Table 8 or Table 17) may optionally be uracil bases (U's),and/or any one or more of the U's may optionally be T's.

b. Oligonucleotide Modifications:

The oligonucleotides described herein may be modified, e.g., comprise amodified sugar moiety, a modified internucleoside linkage, a modifiednucleotide and/or (e.g., and) combinations thereof. In addition, in someembodiments, oligonucleotides may exhibit one or more of the followingproperties: do not mediate alternative splicing; are not immunestimulatory; are nuclease resistant; have improved cell uptake comparedto unmodified oligonucleotides; are not toxic to cells or mammals; haveimproved endosomal exit internally in a cell; minimizes TLR stimulation;or avoid pattern recognition receptors. Any of the modified chemistriesor formats of oligonucleotides described herein can be combined witheach other. For example, one, two, three, four, five, or more differenttypes of modifications can be included within the same oligonucleotide.

In some embodiments, certain nucleotide modifications may be used thatmake an oligonucleotide into which they are incorporated more resistantto nuclease digestion than the native oligodeoxynucleotide oroligoribonucleotide molecules; these modified oligonucleotides surviveintact for a longer time than unmodified oligonucleotides. Specificexamples of modified oligonucleotides include those comprising modifiedbackbones, for example, modified internucleoside linkages such asphosphorothioates, phosphotriesters, methyl phosphonates, short chainalkyl or cycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages. Accordingly, oligonucleotides of thedisclosure can be stabilized against nucleolytic degradation such as bythe incorporation of a modification, e.g., a nucleotide modification.

In some embodiments, an oligonucleotide may be of up to 50 or up to 100nucleotides in length in which 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to18, 2 to 19, 2 to 20, 2 to 25, 2 to 30, 2 to 40, 2 to 45, or morenucleotides of the oligonucleotide are modified nucleotides. Theoligonucleotide may be of 8 to 30 nucleotides in length in which 2 to10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to30 nucleotides of the oligonucleotide are modified nucleotides. Theoligonucleotide may be of 8 to 15 nucleotides in length in which 2 to 4,2 to 5, 2 to 6,2 to 7,2 to 8,2 to 9,2 to 10,2 to 11,2 to 12,2 to 13,2 to14 nucleotides of the oligonucleotide are modified nucleotides.Optionally, the oligonucleotides may have every nucleotide except 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 nucleotides modified. Oligonucleotidemodifications are described further herein.

c. Modified Nucleosides

In some embodiments, the oligonucleotide described herein comprises atleast one nucleoside modified at the 2′ position of the sugar. In someembodiments, an oligonucleotide comprises at least one 2′-modifiednucleoside. In some embodiments, all of the nucleosides in theoligonucleotide are 2′-modified nucleosides.

In some embodiments, the oligonucleotide described herein comprises oneor more non-bicyclic 2′-modified nucleosides, e.g., 2′-deoxy, 2′-fluoro(2′-F), 2′-O-methyl (2′-O-Me), 2′-O-methoxyethyl (2′-MOE),2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA) modified nucleoside.

In some embodiments, the oligonucleotide described herein comprises oneor more 2′-4′ bicyclic nucleosides in which the ribose ring comprises abridge moiety connecting two atoms in the ring, e.g., connecting the2′-O atom to the 4′-C atom via a methylene (LNA) bridge, an ethylene(ENA) bridge, or a (S)-constrained ethyl (cEt) bridge. Examples of LNAsare described in International Patent Application PublicationWO/2008/043753, published on Apr. 17, 2008, and entitled “RNA AntagonistCompounds For The Modulation Of PCSK9”, the contents of which areincorporated herein by reference in its entirety. Examples of ENAs areprovided in International Patent Publication No. WO 2005/042777,published on May 12, 2005, and entitled “APP/ENA Antisense”; Morita etal., Nucleic Acid Res., Suppl 1:241-242, 2001; Surono et al., Hum. GeneTher., 15:749-757, 2004; Koizumi, Curr. Opin. Mol. Ther., 8:144-149,2006 and Horie et al., Nucleic Acids Symp. Ser (Oxf), 49:171-172, 2005;the disclosures of which are incorporated herein by reference in theirentireties. Examples of cEt are provided in U.S. Pat. Nos. 7,101,993;7,399,845 and 7,569,686, each of which is herein incorporated byreference in its entirety.

In some embodiments, the oligonucleotide comprises a modified nucleosidedisclosed in one of the following United States patent or patentApplication Publications: U.S. Pat. No. 7,399,845, issued on Jul. 15,2008, and entitled “6-Modified Bicyclic Nucleic Acid Analogs”; U.S. Pat.No. 7,741,457, issued on Jun. 22, 2010, and entitled “6-ModifiedBicyclic Nucleic Acid Analogs”; U.S. Pat. No. 8,022,193, issued on Sep.20, 2011, and entitled “6-Modified Bicyclic Nucleic Acid Analogs”; U.S.Pat. No. 7,569,686, issued on Aug. 4, 2009, and entitled “Compounds AndMethods For Synthesis Of Bicyclic Nucleic Acid Analogs”; U.S. Pat. No.7,335,765, issued on Feb. 26, 2008, and entitled “Novel Nucleoside AndOligonucleotide Analogues”; U.S. Pat. No. 7,314,923, issued on Jan. 1,2008, and entitled “Novel Nucleoside And Oligonucleotide Analogues”;U.S. Pat. No. 7,816,333, issued on Oct. 19, 2010, and entitled“Oligonucleotide Analogues And Methods Utilizing The Same” and USPublication Number 2011/0009471 now U.S. Pat. No. 8,957,201, issued onFeb. 17, 2015, and entitled “Oligonucleotide Analogues And MethodsUtilizing The Same”, the entire contents of each of which areincorporated herein by reference for all purposes.

In some embodiments, the oligonucleotide comprises at least one modifiednucleoside that results in an increase in Tm of the oligonucleotide in arange of 1° C., 2° C., 3° C., 4° C., or 5° C. compared with anoligonucleotide that does not have the at least one modified nucleoside.The oligonucleotide may have a plurality of modified nucleosides thatresult in a total increase in Tm of the oligonucleotide in a range of 2°C., 3° C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C., 15° C., 20°C., 25° C., 30° C., 35° C., 40° C., 45° C. or more compared with anoligonucleotide that does not have the modified nucleoside.

The oligonucleotide may comprise a mix of nucleosides of differentkinds. For example, an oligonucleotide may comprise a mix of2′-deoxyribonucleosides or ribonucleosides and 2′-fluoro modifiednucleosides. An oligonucleotide may comprise a mix ofdeoxyribonucleosides or ribonucleosides and 2′-O-Me modifiednucleosides. An oligonucleotide may comprise a mix of 2′-fluoro modifiednucleosides and 2′-O-Me modified nucleosides. An oligonucleotide maycomprise a mix of 2′-4′ bicyclic nucleosides and 2′-MOE, 2′-fluoro, or2′-O-Me modified nucleosides. An oligonucleotide may comprise a mix ofnon-bicyclic 2′-modified nucleosides (e.g., 2′-MOE, 2′-fluoro, or2′-O-Me) and 2′-4′ bicyclic nucleosides (e.g., LNA, ENA, cEt).

The oligonucleotide may comprise alternating nucleosides of differentkinds. For example, an oligonucleotide may comprise alternating2′-deoxyribonucleosides or ribonucleosides and 2′-fluoro modifiednucleosides. An oligonucleotide may comprise alternatingdeoxyribonucleosides or ribonucleosides and 2′-O-Me modifiednucleosides. An oligonucleotide may comprise alternating 2′-fluoromodified nucleosides and 2′-O-Me modified nucleosides. Anoligonucleotide may comprise alternating 2′-4′ bicyclic nucleosides and2′-MOE, 2′-fluoro, or 2′-O-Me modified nucleosides. An oligonucleotidemay comprise alternating non-bicyclic 2′-modified nucleosides (e.g.,2′-MOE, 2′-fluoro, or 2′-O-Me) and 2′-4′ bicyclic nucleosides (e.g.,LNA, ENA, cEt).

In some embodiments, an oligonucleotide described herein comprises a5⁻-vinylphosphonate modification, one or more abasic residues, and/orone or more inverted abasic residues.

d. Internucleoside Linkages/Backbones

In some embodiments, oligonucleotide may contain a phosphorothioate orother modified internucleoside linkage. In some embodiments, theoligonucleotide comprises phosphorothioate internucleoside linkages. Insome embodiments, the oligonucleotide comprises phosphorothioateinternucleoside linkages between at least two nucleotides. In someembodiments, the oligonucleotide comprises phosphorothioateinternucleoside linkages between all nucleotides. For example, in someembodiments, oligonucleotides comprise modified internucleoside linkagesat the first, second, and/or (e.g., and) third internucleoside linkageat the 5′ or 3′ end of the nucleotide sequence.

Phosphorus-containing linkages that may be used include, but are notlimited to, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates comprising 3′alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates comprising3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′; see U.S. Pat. Nos. 3,687,808; 4,469,863;4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019;5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111;5,563, 253; 5,571,799; 5,587,361; and 5,625,050.

In some embodiments, oligonucleotides may have heteroatom backbones,such as methylene(methylimino) or MMI backbones; amide backbones (see DeMesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); morpholino backbones(see Summerton and Weller, U.S. Pat. No. 5,034,506); or peptide nucleicacid (PNA) backbones (wherein the phosphodiester backbone of theoligonucleotide is replaced with a polyamide backbone, the nucleotidesbeing bound directly or indirectly to the aza nitrogen atoms of thepolyamide backbone, see Nielsen et al., Science 1991, 254, 1497).

e. Stereospecific Oligonucleotides

In some embodiments, internucleotidic phosphorus atoms ofoligonucleotides are chiral, and the properties of the oligonucleotidesby adjusted based on the configuration of the chiral phosphorus atoms.In some embodiments, appropriate methods may be used to synthesizeP-chiral oligonucleotide analogs in a stereocontrolled manner (e.g., asdescribed in Oka N, Wada T, Stereocontrolled synthesis ofoligonucleotide analogs containing chiral internucleotidic phosphorusatoms. Chem Soc Rev. 2011 December; 40(12):5829-43.) In someembodiments, phosphorothioate containing oligonucleotides comprisenucleoside units that are joined together by either substantially all Spor substantially all Rp phosphorothioate intersugar linkages areprovided. In some embodiments, such phosphorothioate oligonucleotideshaving substantially chirally pure intersugar linkages are prepared byenzymatic or chemical synthesis, as described, for example, in U.S. Pat.No. 5,587,261, issued on Dec. 12, 1996, the contents of which areincorporated herein by reference in their entirety. In some embodiments,chirally controlled oligonucleotides provide selective cleavage patternsof a target nucleic acid. For example, in some embodiments, a chirallycontrolled oligonucleotide provides single site cleavage within acomplementary sequence of a nucleic acid, as described, for example, inUS Patent Application Publication 20170037399 Al, published on Feb. 2,2017, entitled “CHIRAL DESIGN”, the contents of which are incorporatedherein by reference in their entirety.

f. Morpholinos

In some embodiments, the oligonucleotide may be a morpholino-basedcompounds. Morpholino-based oligomeric compounds are described in DwaineA. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510);Genesis, volume 30, issue 3, 2001; Heasman, J., Dev. Biol., 2002, 243,209-214; Nasevicius et al., Nat. Genet., 2000, 26, 216-220; Lacerra etal., Proc. Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No.5,034,506, issued Jul. 23, 1991. In some embodiments, themorpholino-based oligomeric compound is a phosphorodiamidate morpholinooligomer (PMO) (e.g., as described in Iverson, Curr. Opin. Mol. Ther.,3:235-238, 2001; and Wang et al., J. Gene Med., 12:354-364, 2010; thedisclosures of which are incorporated herein by reference in theirentireties).

g. Peptide Nucleic Acids (PNAs)

In some embodiments, both a sugar and an internucleoside linkage (thebackbone) of the nucleotide units of an oligonucleotide are replacedwith novel groups. In some embodiments, the base units are maintainedfor hybridization with an appropriate nucleic acid target compound. Onesuch oligomeric compound, an oligonucleotide mimetic that has been shownto have excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, forexample, an aminoethylglycine backbone. The nucleobases are retained andare bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative publication that report thepreparation of PNA compounds include, but are not limited to, U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

h. Gapmers

In some embodiments, an oligonucleotide described herein is a gapmer. Agapmer oligonucleotide generally has the formula 5′-X—Y—Z-3′, with X andZ as flanking regions around a gap region Y. In some embodiments,flanking region X of formula 5′-X—Y—Z-3′ is also referred to as Xregion, flanking sequence X, 5′ wing region X, or 5′ wing segment. Insome embodiments, flanking region Z of formula 5′-X—Y—Z-3′ is alsoreferred to as Z region, flanking sequence Z, 3′ wing region Z, or 3′wing segment. In some embodiments, gap region Y of formula 5′-X—Y—Z-3′is also referred to as Y region, Y segment, or gap-segment Y. In someembodiments, each nucleoside in the gap region Y is a2′-deoxyribonucleoside, and neither the 5′ wing region X or the 3′ wingregion Z contains any 2′-deoxyribonucleosides.

In some embodiments, the Y region is a contiguous stretch ofnucleotides, e.g., a region of 6 or more DNA nucleotides, which arecapable of recruiting an RNAse, such as RNAse H. In some embodiments,the gapmer binds to the target nucleic acid, at which point an RNAse isrecruited and can then cleave the target nucleic acid. In someembodiments, the Y region is flanked both 5′ and 3′ by regions X and Zcomprising high-affinity modified nucleosides, e.g., one to sixhigh-affinity modified nucleosides. Examples of high affinity modifiednucleosides include, but are not limited to, 2′-modified nucleosides(e.g., 2′-MOE, 2′O-Me, 2′-F) or 2′-4′ bicyclic nucleosides (e.g., LNA,cEt, ENA). In some embodiments, the flanking sequences X and Z may be of1-20 nucleotides, 1-8 nucleotides, or 1-5 nucleotides in length. Theflanking sequences X and Z may be of similar length or of dissimilarlengths. In some embodiments, the gap-segment Y may be a nucleotidesequence of 5-20 nucleotides, 5-15 twelve nucleotides, or 6-10nucleotides in length.

In some embodiments, the gap region of the gapmer oligonucleotides maycontain modified nucleotides known to be acceptable for efficient RNaseH action in addition to DNA nucleotides, such as C4′-substitutednucleotides, acyclic nucleotides, and arabino-configured nucleotides. Insome embodiments, the gap region comprises one or more unmodifiedinternucleoside linkages. In some embodiments, one or both flankingregions each independently comprise one or more phosphorothioateinternucleoside linkages (e.g., phosphorothioate internucleosidelinkages or other linkages) between at least two, at least three, atleast four, at least five or more nucleotides. In some embodiments, thegap region and two flanking regions each independently comprise modifiedinternucleoside linkages (e.g., phosphorothioate internucleosidelinkages or other linkages) between at least two, at least three, atleast four, at least five or more nucleotides.

A gapmer may be produced using appropriate methods. Representative U.S.patents, U.S. patent publications, and PCT publications that teach thepreparation of gapmers include, but are not limited to, U.S. Pat. Nos.5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; 5,700,922;5,898,031; 7,015,315; 7,101,993; 7,399,845; 7,432,250; 7,569,686;7,683,036; 7,750,131; 8,580,756; 9,045,754; 9,428,534; 9,695,418;10,017,764; 10,260,069; 9,428,534; 8,580,756; U.S. patent publicationNos. US20050074801, US20090221685; US20090286969, US20100197762, andUS20110112170; PCT publication Nos. WO2004069991; WO2005023825;WO2008049085 and WO2009090182; and EP Patent No. EP2,149,605, each ofwhich is herein incorporated by reference in its entirety.

In some embodiments, a gapmer is 10-40 nucleosides in length. Forexample, a gapmer may be 10-40, 10-35, 10-30, 10-25, 10-20, 10-15,15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40,25-35, 25-30, 30-40, 30-35, or 35-40 nucleosides in length. In someembodiments, a gapmer is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,or 40 nucleosides in length.

In some embodiments, the gap region Y in a gapmer is 5-20 nucleosides inlength. For example, the gap region Y may be 5-20, 5-15, 5-10, 10-20,10-15, or 15-20 nucleosides in length. In some embodiments, the gapregion Y is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20nucleosides in length. In some embodiments, each nucleoside in the gapregion Y is a 2′-deoxyribonucleoside. In some embodiments, allnucleosides in the gap region Y are 2′-deoxyribonucleosides. In someembodiments, one or more of the nucleosides in the gap region Y is amodified nucleoside (e.g., a 2′ modified nucleoside such as thosedescribed herein). In some embodiments, one or more cytosines in the gapregion Y are optionally 5-methyl-cytosines. In some embodiments, eachcytosine in the gap region Y is a 5-methyl-cytosines.

In some embodiments, the 5′wing region of a gapmer (X in the 5′-X—Y—Z-3′formula) and the 3′wing region of a gapmer (Z in the 5′-X—Y—Z-3′formula) are independently 1-20 nucleosides long. For example, the5′wing region of a gapmer (X in the 5′-X—Y—Z-3′ formula) and the 3′wingregion of the gapmer (Z in the 5′-X—Y—Z-3′ formula) may be independently1-20, 1-15, 1-10, 1-7, 1-5, 1-3, 1-2, 2-5, 2-7, 3-5, 3-7, 5-20, 5-15,5-10, 10-20, 10-15, or 15-20 nucleosides long. In some embodiments, the5′wing region of the gapmer (X in the 5′-X—Y—Z-3′ formula) and the3′wing region of the gapmer (Z in the 5′-X—Y—Z-3′ formula) areindependently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20 nucleosides long. In some embodiments, the 5′wing regionof the gapmer (X in the 5′-X—Y—Z-3′ formula) and the 3′wing region ofthe gapmer (Z in the 5′-X—Y—Z-3′ formula) are of the same length. Insome embodiments, the 5′wing region of the gapmer (X in the 5′-X—Y—Z-3′formula) and the 3′wing region of the gapmer (Z in the 5′-X—Y—Z-3′formula) are of different lengths. In some embodiments, the 5′wingregion of the gapmer (X in the 5′-X—Y—Z-3′ formula) is longer than the3′wing region of the gapmer (Z in the 5′-X—Y—Z-3′ formula). In someembodiments, the 5′wing region of the gapmer (X in the 5′-X—Y—Z-3′formula) is shorter than the 3′wing region of the gapmer (Z in the5′-X—Y—Z-3′ formula).

In some embodiments, a gapmer comprises a 5′-X—Y—Z-3′ of 5-10-5, 4-12-4,3-14-3, 2-16-2, 1-18-1, 3-10-3, 2-10-2, 1-10-1, 2-8-2, 4-6-4, 3-6-3,2-6-2, 4-7-4, 3-7-3, 2-7-2, 4-8-4, 3-8-3, 2-8-2, 1-8-1, 2-9-2, 1-9-1,2-10-2, 1-10-1, 1-12-1, 1-16-1, 2-15-1, 1-15-2, 1-14-3, 3-14-1, 2-14-2,1-13-4, 4-13-1, 2-13-3, 3-13-2, 1-12-5, 5-12-1, 2-12-4, 4-12-2, 3-12-3,1-11-6, 6- 11-1, 2-11-5, 5-11-2, 3-11-4, 4-11-3, 1-17-1, 2-16-1, 1-16-2,1-15-3, 3-15-1, 2-15-2, 1-14-4, 4-14-1, 2-14-3, 3-14-2, 1-13-5, 5-13-1,2-13-4, 4-13-2, 3-13-3, 1-12-6, 6-12-1, 2-12-5, 5-12-2, 3-12-4, 4-12-3,1-11-7, 7-11-1, 2-11-6, 6-11-2, 3-11-5, 5-11-3, 4-11-4, 1-18-1, 1-17-2,2-17-1, 1-16-3, 1-16-3, 2-16-2, 1-15-4, 4-15-1, 2-15-3, 3-15-2, 1-14-5,5-14-1, 2-14-4, 4-14-2, 3-14-3, 1-13-6, 6-13-1, 2-13-5, 5-13-2, 3-13-4,4-13-3, 1-12-7, 7-12-1, 2-12-6, 6-12-2, 3-12-5, 5-12-3, 1-11-8, 8-11-1,2-11-7, 7-11-2, 3-11-6, 6-11-3, 4-11-5, 5-11-4, 1-18-1, 1-17-2, 2-17-1,1-16-3, 3-16-1, 2-16-2, 1-15-4, 4-15-1, 2-15-3, 3-15-2, 1-14-5, 2-14-4,4-14-2, 3-14-3, 1-13-6, 6-13-1, 2-13-5, 5-13-2, 3-13-4, 4-13-3, 1-12-7,7-12-1, 2-12-6, 6-12-2, 3-12-5, 5-12-3, 1-11-8, 8-11-1, 2-11-7, 7-11-2,3-11-6, 6-11-3, 4-11-5, 5-11-4, 1-19-1, 1-18-2, 2-18-1, 1-17-3, 3-17-1,2-17-2, 1-16-4, 4-16-1, 2-16-3, 3-16-2, 1-15-5, 2-15-4, 4-15-2, 3-15-3,1-14-6, 6-14-1, 2-14-5, 5-14-2, 3-14-4, 4-14-3, 1-13-7, 7-13-1, 2-13-6,6-13-2, 3-13-5, 5-13-3, 4-13-4, 1-12-8, 8-12-1, 2-12-7, 7-12-2, 3-12-6,6-12-3, 4-12-5, 5-12-4, 2-11-8, 8-11-2, 3-11-7, 7-11-3, 4-11-6, 6-11-4,5-11-5, 1-20-1, 1-19-2, 2-19-1, 1-18-3, 3-18-1, 2-18-2, 1-17-4, 4-17-1,2-17-3, 3-17-2, 1-16-5, 2-16-4, 4-16-2, 3-16-3, 1-15-6, 6-15-1, 2-15-5,5-15-2, 3-15-4, 4-15-3, 1-14-7, 7-14-1, 2-14-6, 6-14-2, 3-14-5, 5-14-3,4-14-4, 1-13-8, 8-13-1, 2-13-7, 7-13-2, 3-13-6, 6-13-3, 4-13-5, 5-13-4,2-12-8, 8-12-2, 3-12-7, 7-12-3, 4-12-6, 6-12-4, 5-12-5, 3-11-8, 8-11-3,4-11-7, 7-11-4, 5-11-6, 6-11-5, 1-21-1, 1-20-2, 2-20-1, 1-20-3, 3-19-1,2-19-2, 1-18-4, 4-18-1, 2-18-3, 3-18-2, 1-17-5, 2-17-4, 4-17-2, 3-17-3,1-16-6, 6-16-1, 2-16-5, 5-16-2, 3-16-4, 4-16-3, 1-15-7, 7-15-1, 2-15-6,6-15-2, 3-15-5, 5-15-3, 4-15-4, 1-14-8, 8-14-1, 2-14-7, 7-14-2, 3-14-6,6-14-3, 4-14-5, 5-14-4, 2-13-8, 8-13-2, 3-13-7, 7-13-3, 4-13-6, 6-13-4,5-13-5, 1-12-10, 10-12-1, 2-12-9, 9-12-2, 3-12-8, 8-12-3, 4-12-7,7-12-4, 5-12-6, 6-12-5, 4-11-8, 8-11-4, 5-11-7, 7-11-5, 6-11-6, 1-22-1,1-21-2, 2-21-1, 1-21-3, 3-20-1, 2-20-2, 1-19-4, 4-19-1, 2-19-3, 3-19-2,1-18-5, 2-18-4, 4-18-2, 3-18-3, 1-17-6, 6-17-1, 2-17-5, 5-17-2, 3-17-4,4-17-3, 1-16-7, 7-16-1, 2-16-6, 6-16-2, 3-16-5, 5-16-3, 4-16-4, 1-15-8,8-15-1, 2-15-7, 7-15-2, 3-15-6, 6-15-3, 4-15-5, 5-15-4, 2-14-8, 8-14-2,3-14-7, 7-14-3, 4-14-6, 6-14-4, 5-14-5, 3-13-8, 8-13-3, 4-13-7, 7-13-4,5-13-6, 6-13-5, 4-12-8, 8-12-4, 5-12-7, 7-12-5, 6-12-6, 5-11-8, 8-11-5,6-11-7, or 7-11-6. The numbers indicate the number of nucleosides in X,Y, and Z regions in the 5′-X—Y—Z-3′ gapmer.

In some embodiments, one or more nucleosides in the 5′wing region of agapmer (X in the 5′-X—Y—Z-3′ formula) or the 3′wing region of a gapmer(Z in the 5′-X—Y—Z-3′ formula) are modified nucleotides (e.g.,high-affinity modified nucleosides). In some embodiments, the modifiednucleoside (e.g., high-affinity modified nucleosides) is a 2′-modifiednucleoside. In some embodiments, the 2′-modified nucleoside is a 2′-4′bicyclic nucleoside or a non-bicyclic 2′-modified nucleoside. In someembodiments, the high-affinity modified nucleoside is a 2′-4′ bicyclicnucleoside (e.g., LNA, cEt, or ENA) or a non-bicyclic 2′-modifiednucleoside (e.g., 2′-fluoro (2′-F), 2′-O-methyl (2′-O-Me),2′-O-methoxyethyl (2′-MO E), 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or2′-O—N-methylacetamido (2′-O-NMA)).

In some embodiments, one or more nucleosides in the 5′wing region of agapmer (X in the 5′-X—Y—Z-3′ formula) are high-affinity modifiednucleosides. In some embodiments, each nucleoside in the 5′wing regionof the gapmer (X in the 5′-X—Y—Z-3′ formula) is a high-affinity modifiednucleoside. In some embodiments, one or more nucleosides in the 3′wingregion of a gapmer (Z in the 5′-X—Y—Z-3′ formula) are high-affinitymodified nucleosides. In some embodiments, each nucleoside in the 3′wingregion of the gapmer (Z in the 5′-X—Y—Z-3′ formula) is a high-affinitymodified nucleoside. In some embodiments, one or more nucleosides in the5′wing region of the gapmer (X in the 5′-X—Y—Z-3′ formula) arehigh-affinity modified nucleosides and one or more nucleosides in the3′wing region of the gapmer (Z in the 5′-X—Y—Z-3′ formula) arehigh-affinity modified nucleosides. In some embodiments, each nucleosidein the 5′wing region of the gapmer (X in the 5′-X—Y—Z-3′ formula) is ahigh-affinity modified nucleoside and each nucleoside in the 3′wingregion of the gapmer (Z in the 5′-X—Y—Z-3′ formula) is high-affinitymodified nucleoside.

In some embodiments, the 5′wing region of a gapmer (X in the 5′-X—Y—Z-3′formula) comprises the same high affinity nucleosides as the 3′wingregion of the gapmer (Z in the 5′-X—Y—Z-3′ formula). For example, the5′wing region of the gapmer (X in the 5′-X—Y—Z-3′ formula) and the3′wing region of the gapmer (Z in the 5′-X—Y—Z-3′ formula) may compriseone or more non-bicyclic 2′-modified nucleosides (e.g., 2′-MOE or2′-O-Me). In another example, the 5′wing region of the gapmer (X in the5′-X—Y—Z-3′ formula) and the 3′wing region of the gapmer (Z in the5′-X—Y—Z-3′ formula) may comprise one or more 2′-4′ bicyclic nucleosides(e.g., LNA or cEt). In some embodiments, each nucleoside in the 5′wingregion of the gapmer (X in the 5′-X—Y—Z-3′ formula) and the 3′wingregion of the gapmer (Z in the 5′-X—Y—Z-3′ formula) is a non-bicyclic2′-modified nucleosides (e.g., 2′-MOE or 2′-O-Me). In some embodiments,each nucleoside in the 5′wing region of the gapmer (X in the 5′-X—Y—Z-3′formula) and the 3′wing region of the gapmer (Z in the 5′-X—Y—Z-3′formula) is a 2′-4′ bicyclic nucleosides (e.g., LNA or cEt).

In some embodiments, a gapmer comprises a 5′-X—Y—Z-3′ configuration,wherein X and Z is independently 1-7 (e.g., 1, 2, 3, 4, 5, 6, or 7)nucleosides in length and Y is 6-10 (e.g., 6, 7, 8, 9, or 10)nucleosides in length, wherein each nucleoside in X and Z is anon-bicyclic 2′-modified nucleosides (e.g., 2′-MOE or 2′-O-Me) and eachnucleoside in Y is a 2′-deoxyribonucleoside. In some embodiments, thegapmer comprises a 5′-X—Y—Z-3′ configuration, wherein X and Z isindependently 1-7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides in lengthand Y is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, whereineach nucleoside in X and Z is a 2′-4′ bicyclic nucleosides (e.g., LNA orcEt) and each nucleoside in Y is a 2′-deoxyribonucleoside. In someembodiments, the 5′wing region of the gapmer (X in the 5′-X—Y—Z-3′formula) comprises different high affinity nucleosides as the 3′wingregion of the gapmer (Z in the 5′-X—Y—Z-3′ formula). For example, the5′wing region of the gapmer (X in the 5′-X—Y—Z-3′ formula) may compriseone or more non-bicyclic 2′-modified nucleosides (e.g., 2′-MOE or2′-O-Me) and the 3′wing region of the gapmer (Z in the 5′-X—Y—Z-3′formula) may comprise one or more 2′-4′ bicyclic nucleosides (e.g., LNAor cEt). In another example, the 3′wing region of the gapmer (Z in the5′-X—Y—Z-3′ formula) may comprise one or more non-bicyclic 2′-modifiednucleosides (e.g., 2′-MOE or 2′-O-Me) and the 5′wing region of thegapmer (X in the 5′-X—Y—Z-3′ formula) may comprise one or more 2′-4′bicyclic nucleosides (e.g., LNA or cEt).

In some embodiments, a gapmer comprises a 5′-X—Y—Z-3′ configuration,wherein X and Z is independently 1-7 (e.g., 1, 2, 3, 4, 5, 6, or 7)nucleosides in length and Y is 6-10 (e.g., 6, 7, 8, 9, or 10)nucleosides in length, wherein each nucleoside in X is a non-bicyclic2′-modified nucleosides (e.g., 2′-MOE or 2′-O-Me), each nucleoside in Zis a 2′-4′ bicyclic nucleosides (e.g., LNA or cEt), and each nucleosidein Y is a 2′-deoxyribonucleoside. In some embodiments, the gapmercomprises a 5′-X—Y—Z-3′ configuration, wherein X and Z is independently1-7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6-10(e.g., 6, 7, 8, 9, or 10) nucleosides in length, wherein each nucleosidein X is a 2′-4′ bicyclic nucleosides (e.g., LNA or cEt), each nucleosidein Z is a non-bicyclic 2′-modified nucleosides (e.g., 2′-MOE or 2′-O-Me)and each nucleoside in Y is a 2′-deoxyribonucleoside.

In some embodiments, the 5′wing region of a gapmer (X in the 5′-X—Y—Z-3′formula) comprises one or more non-bicyclic 2′-modified nucleosides(e.g., 2′-MOE or 2′-O-Me) and one or more 2′-4′ bicyclic nucleosides(e.g., LNA or cEt). In some embodiments, the 3′wing region of the gapmer(Z in the 5′-X—Y—Z-3′ formula) comprises one or more non-bicyclic2′-modified nucleosides (e.g., 2′-MOE or 2′-O-Me) and one or more 2′-4′bicyclic nucleosides (e.g., LNA or cEt). In some embodiments, both the5′wing region of the gapmer (X in the 5′-X—Y—Z-3′ formula) and the3′wing region of the gapmer (Z in the 5′-X—Y—Z-3′ formula) comprise oneor more non-bicyclic 2′-modified nucleosides (e.g., 2′-MOE or 2′-O-Me)and one or more 2′-4′ bicyclic nucleosides (e.g., LNA or cEt).

In some embodiments, a gapmer comprises a 5′-X—Y—Z-3′ configuration,wherein X and Z is independently 2-7 (e.g., 2, 3, 4, 5, 6, or 7)nucleosides in length and Y is 6-10 (e.g., 6, 7, 8, 9, or 10)nucleosides in length, wherein at least one but not all (e.g., 1, 2, 3,4, 5, or 6) of positions 1, 2, 3, 4, 5, 6, or 7 in X (the 5′ mostposition is position 1) is a non-bicyclic 2′-modified nucleoside (e.g.,2′-MOE or 2′-O-Me), wherein the rest of the nucleosides in both X and Zare 2′-4′ bicyclic nucleosides (e.g., LNA or cEt), and wherein eachnucleoside in Y is a 2′deoxyribonucleoside. In some embodiments, thegapmer comprises a 5′-X—Y—Z-3′ configuration, wherein X and Z isindependently 2-7 (e.g., 2, 3, 4, 5, 6, or 7) nucleosides in length andY is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, wherein atleast one but not all (e.g., 1, 2, 3, 4, 5, or 6) of positions 1, 2, 3,4, 5, 6, or 7 in Z (the 5′ most position is position 1) is anon-bicyclic 2′-modified nucleoside (e.g., 2′-MOE or 2′-O-Me), whereinthe rest of the nucleosides in both X and Z are 2′-4′ bicyclicnucleosides (e.g., LNA or cEt), and wherein each nucleoside in Y is a2′deoxyribonucleoside. In some embodiments, the gapmer comprises a5′-X—Y—Z-3′ configuration, wherein X and Z is independently 2-7 (e.g.,2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6-10 (e.g., 6, 7, 8,9, or 10) nucleosides in length, wherein at least one but not all (e.g.,1, 2, 3, 4, 5, or 6) of positions 1, 2, 3, 4, 5, 6, or 7 in X and atleast one of positions but not all (e.g., 1, 2, 3, 4, 5, or 6) 1, 2, 3,4, 5, 6, or 7 in Z (the 5′ most position is position 1) is anon-bicyclic 2′-modified nucleoside (e.g., 2′-MOE or 2′-O-Me), whereinthe rest of the nucleosides in both X and Z are 2′-4′ bicyclicnucleosides (e.g., LNA or cEt), and wherein each nucleoside in Y is a2′deoxyribonucleoside.

Non-limiting examples of gapmers configurations with a mix ofnon-bicyclic 2′-modified nucleoside (e.g., 2′-MOE or 2′-O-Me) and 2′-4′bicyclic nucleosides (e.g., LNA or cEt) in the 5′wing region of thegapmer (X in the 5′-X—Y—Z-3′ formula) and/or the 3′wing region of thegapmer (Z in the 5′-X—Y—Z-3′ formula) include: BBB-(D)n-BBBAA;KKK-(D)n-KKKAA; LLL-(D)n-LLLAA; BBB-(D)n-BBBEE; KKK-(D)n-KKKEE;LLL-(D)n-LLLEE; BBB-(D)n-BBBAA; KKK-(D)n-KKKAA; LLL-(D)n-LLLAA;BBB-(D)n-BBBEE; KKK-(D)n-KKKEE; LLL-(D)n-LLLEE; BBB-(D)n-BBBAAA;KKK-(D)n-KKKAAA; LLL-(D)n-LLLAAA; BBB-(D)n-BBBEEE; KKK-(D)n-KKKEEE;LLL-(D)n-LLLEEE; BBB-(D)n-BBBAAA; KKK-(D)n-KKKAAA; LLL-(D)n-LLLAAA;BBB-(D)n-BBBEEE; KKK-(D)n-KKKEEE; LLL-(D)n-LLLEEE; BABA-(D)n-ABAB;KAKA-(D)n-AKAK; LALA-(D)n-ALAL; BEBE-(D)n-EBEB; KEKE-(D)n-EKEK;LELE-(D)n-ELEL; BABA-(D)n-ABAB; KAKA-(D)n-AKAK; LALA-(D)n-ALAL;BEBE-(D)n-EBEB; KEKE-(D)n-EKEK; LELE-(D)n-ELEL; ABAB-(D)n-ABAB;AKAK-(D)n-AKAK; ALAL-(D)n-ALAL; EBEB-(D)n-EBEB; EKEK-(D)n-EKEK;ELEL-(D)n-ELEL; ABAB-(D)n-ABAB; AKAK-(D)n-AKAK; ALAL-(D)n-ALAL;EBEB-(D)n-EBEB; EKEK-(D)n-EKEK; ELEL-(D)n-ELEL; AABB-(D)n-BBAA;BBAA-(D)n-AABB; AAKK-(D)n-KKAA; AALL-(D)n-LLAA; EEBB-(D)n-BBEE;EEKK-(D)n-KKEE; EELL-(D)n-LLEE; AABB-(D)n-BBAA; AAKK-(D)n-KKAA;AALL-(D)n-LLAA; EEBB-(D)n-BBEE; EEKK-(D)n-KKEE; EELL-(D)n-LLEE;BBB-(D)n-BBA; KKK-(D)n-KKA; LLL-(D)n-LLA; BBB-(D)n-BBE; KKK-(D)n-KKE;LLL-(D)n-LLE; BBB-(D)n-BBA; KKK-(D)n-KKA; LLL-(D)n-LLA; BBB-(D)n-BBE;KKK-(D)n-KKE; LLL-(D)n-LLE; BBB-(D)n-BBA; KKK-(D)n-KKA; LLL-(D)n-LLA;BBB-(D)n-BBE; KKK-(D)n-KKE; LLL-(D)n-LLE; ABBB-(D)n-BBBA;AKKK-(D)n-KKKA; ALLL-(D)n-LLLA; EBBB-(D)n-BBBE; EKKK-(D)n-KKKE;ELLL-(D)n-LLLE; ABBB-(D)n-BBBA; AKKK-(D)n-KKKA; ALLL-(D)n-LLLA;EBBB-(D)n-BBBE; EKKK-(D)n-KKKE; ELLL-(D)n-LLLE; ABBB-(D)n-BBBAA;AKKK-(D)n-KKKAA; ALLL-(D)n-LLLAA; EBBB-(D)n-BBBEE; EKKK-(D)n-KKKEE;ELLL-(D)n-LLLEE; ABBB-(D)n-BBBAA; AKKK-(D)n-KKKAA; ALLL-(D)n-LLLAA;EBBB-(D)n-BBBEE; EKKK-(D)n-KKKEE; ELLL-(D)n-LLLEE; AABBB-(D)n-BBB;AAKKK-(D)n-KKK; AALLL-(D)n-LLL; EEBBB-(D)n-BBB; EEKKK-(D)n-KKK;EELLL-(D)n-LLL; AABBB-(D)n-BBB; AAKKK-(D)n-KKK; AALLL-(D)n-LLL;EEBBB-(D)n-BBB; EEKKK-(D)n-KKK; EELLL-(D)n-LLL; AABBB-(D)n-BBBA;AAKKK-(D)n-KKKA; AALLL-(D)n-LLLA; EEBBB-(D)n-BBBE; EEKKK-(D)n-KKKE;EELLL-(D)n-LLLE; AABBB-(D)n-BBBA; AAKKK-(D)n-KKKA; AALLL-(D)n-LLLA;EEBBB-(D)n-BBBE; EEKKK-(D)n-KKKE; EELLL-(D)n-LLLE; ABBAABB-(D)n-BB;AKKAAKK-(D)n-KK; ALLAALLL-(D)n-LL; EBBEEBB-(D)n-BB; EKKEEKK-(D)n-KK;ELLEELL-(D)n-LL; ABBAABB-(D)n-BB; AKKAAKK-(D)n-KK; ALLAALL-(D)n-LL;EBBEEBB-(D)n-BB; EKKEEKK-(D)n-KK; ELLEELL-(D)n-LL; ABBABB-(D)n-BBB;AKKAKK-(D)n-KKK; ALLALLL-(D)n-LLL; EBBEBB-(D)n-BBB; EKKEKK-(D)n-KKK;ELLELL-(D)n-LLL; ABBABB-(D)n-BBB; AKKAKK-(D)n-KKK; ALLALL-(D)n-LLL;EBBEBB-(D)n-BBB; EKKEKK-(D)n-KKK; ELLELL-(D)n-LLL; EEEK-(D)n-EEEEEEEE;EEK-(D)n-EEEEEEEEE; EK-(D)n-EEEEEEEEEE; EK-(D)n-EEEKK; K-(D)n-EEEKEKE;K-(D)n-EEEKEKEE; K-(D)n-EEKEK; EK-(D)n-EEEEKEKE; EK-(D)n-EEEKEK;EEK-(D)n-KEEKE; EK-(D)n-EEKEK; EK-(D)n-KEEK; EEK-(D)n-EEEKEK;EK-(D)n-KEEEKEE; EK-(D)n-EEKEKE; EK-(D)n-EEEKEKE; and EK-(D)n-EEEEKEK;“A” nucleosides comprise a 2′-modified nucleoside; “B” represents a2′-4′ bicyclic nucleoside; “K” represents a constrained ethyl nucleoside(cEt); “L” represents an LNA nucleoside; and “E” represents a 2′-MOEmodified ribonucleoside; “D” represents a 2′-deoxyribonucleoside; “n”represents the length of the gap segment (Y in the 5′-X—Y—Z-3′configuration) and is an integer between 1-20.

In some embodiments, any one of the gapmers described herein comprisesone or more modified nucleoside linkages (e.g., a phosphorothioatelinkage) in each of the X, Y, and Z regions. In some embodiments, eachinternucleoside linkage in the any one of the gapmers described hereinis a phosphorothioate linkage. In some embodiments, each of the X, Y,and Z regions independently comprises a mix of phosphorothioate linkagesand phosphodiester linkages. In some embodiments, each internucleosidelinkage in the gap region Y is a phosphorothioate linkage, the 5′wingregion X comprises a mix of phosphorothioate linkages and phosphodiesterlinkages, and the 3′wing region Z comprises a mix of phosphorothioatelinkages and phosphodiester linkages.

i. Mixmers

In some embodiments, an oligonucleotide described herein may be a mixmeror comprise a mixmer sequence pattern. In general, mixmers areoligonucleotides that comprise both naturally and non-naturallyoccurring nucleosides or comprise two different types of non-naturallyoccurring nucleosides typically in an alternating pattern. Mixmersgenerally have higher binding affinity than unmodified oligonucleotidesand may be used to specifically bind a target molecule, e.g., to block abinding site on the target molecule. Generally, mixmers do not recruitan RNase to the target molecule and thus do not promote cleavage of thetarget molecule. Such oligonucleotides that are incapable of recruitingRNase H have been described, for example, see WO2007/112754 orWO2007/112753.

In some embodiments, the mixmer comprises or consists of a repeatingpattern of nucleoside analogues and naturally occurring nucleosides, orone type of nucleoside analogue and a second type of nucleosideanalogue. However, a mixmer need not comprise a repeating pattern andmay instead comprise any arrangement of modified nucleoside s andnaturally occurring nucleoside s or any arrangement of one type ofmodified nucleoside and a second type of modified nucleoside. Therepeating pattern, may, for instance be every second or every thirdnucleoside is a modified nucleoside, such as LNA, and the remainingnucleoside s are naturally occurring nucleosides, such as DNA, or are a2′ substituted nucleoside analogue such as 2′-MOE or 2′ fluoroanalogues, or any other modified nucleoside described herein. It isrecognized that the repeating pattern of modified nucleoside, such asLNA units, may be combined with modified nucleoside at fixedpositions—e.g. at the 5′ or 3′ termini.

In some embodiments, a mixmer does not comprise a region of more than 5,more than 4, more than 3, or more than 2 consecutive naturally occurringnucleosides, such as DNA nucleosides. In some embodiments, the mixmercomprises at least a region consisting of at least two consecutivemodified nucleoside, such as at least two consecutive LNAs. In someembodiments, the mixmer comprises at least a region consisting of atleast three consecutive modified nucleoside units, such as at leastthree consecutive LNAs.

In some embodiments, the mixmer does not comprise a region of more than7, more than 6, more than 5, more than 4, more than 3, or more than 2consecutive nucleoside analogues, such as LNAs. In some embodiments, LNAunits may be replaced with other nucleoside analogues, such as thosereferred to herein.

Mixmers may be designed to comprise a mixture of affinity enhancingmodified nucleosides, such as in non-limiting example LNA nucleosidesand 2′-O-Me nucleosides. In some embodiments, a mixmer comprisesmodified internucleoside linkages (e.g., phosphorothioateinternucleoside linkages or other linkages) between at least two, atleast three, at least four, at least five or more nucleosides.

A mixmer may be produced using any suitable method. Representative U.S.patents, U.S. patent publications, and PCT publications that teach thepreparation of mixmers include U.S. patent publication Nos.US20060128646, US20090209748, US20090298916, US20110077288, andUS20120322851, and U.S. Pat. No. 7,687,617.

In some embodiments, a mixmer comprises one or more morpholinonucleosides. For example, in some embodiments, a mixmer may comprisemorpholino nucleosides mixed (e.g., in an alternating manner) with oneor more other nucleosides (e.g., DNA, RNA nucleosides) or modifiednucleosides (e.g., LNA, 2′-O-Me nucleosides).

In some embodiments, mixmers are useful for splice correcting or exonskipping, for example, as reported in Touznik A., et al., LNA/DNAmixmer-based antisense oligonucleotides correct alternative splicing ofthe SMN2 gene and restore SMN protein expression in type 1 SMAfibroblasts Scientific Reports, volume 7, Article number: 3672 (2017),Chen S. et al., Synthesis of a Morpholino Nucleic Acid (MNA)-UridinePhosphoramidite, and Exon Skipping Using MNA/2′-O-Methyl MixmerAntisense Oligonucleotide, Molecules 2016, 21, 1582, the contents ofeach which are incorporated herein by reference.

j. RNA Interference (RNAi)

In some embodiments, oligonucleotides provided herein may be in the formof small interfering RNAs (siRNA), also known as short interfering RNAor silencing RNA. SiRNA, is a class of double-stranded RNA molecules,typically about 20-25 base pairs in length that target nucleic acids(e.g., mRNAs) for degradation via the RNA interference (RNAi) pathway incells. Specificity of siRNA molecules may be determined by the bindingof the antisense strand of the molecule to its target RNA. EffectivesiRNA molecules are generally less than 30 to 35 base pairs in length toprevent the triggering of non-specific RNA interference pathways in thecell via the interferon response, although longer siRNA can also beeffective. In some embodiments, the siRNA molecules are 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 35, 40, 45, 50, or more base pairs in length. In some embodiments,the siRNA molecules are 8 to 30 base pairs in length, 10 to 15 basepairs in length, 10 to 20 base pairs in length, 15 to 25 base pairs inlength, 19 to 21 base pairs in length, 21 to 23 base pairs in length.

Following selection of an appropriate target RNA sequence, siRNAmolecules that comprise a nucleotide sequence complementary to all or aportion of the target sequence, i.e. an antisense sequence, can bedesigned and prepared using appropriate methods (see, e.g., PCTPublication Number WO 2004/016735; and U.S. Patent Publication Nos.2004/0077574 and 2008/0081791). The siRNA molecule can be doublestranded (i.e. a dsRNA molecule comprising an antisense strand and acomplementary sense strand that hybridizes to form the dsRNA) orsingle-stranded (i.e. a ssRNA molecule comprising just an antisensestrand). The siRNA molecules can comprise a duplex, asymmetric duplex,hairpin or asymmetric hairpin secondary structure, havingself-complementary sense and antisense strands.

In some embodiments, the antisense strand of the siRNA molecule is 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, or more nucleotides in length. In someembodiments, the antisense strand is 8 to 50 nucleotides in length, 8 to40 nucleotides in length, 8 to 30 nucleotides in length, 10 to 15nucleotides in length, 10 to 20 nucleotides in length, 15 to 25nucleotides in length, 19 to 21 nucleotides in length, 21 to 23nucleotides in lengths.

In some embodiments, the sense strand of the siRNA molecule is 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 35, 40, 45, 50, or more nucleotides in length. In someembodiments, the sense strand is 8 to 50 nucleotides in length, 8 to 40nucleotides in length, 8 to 30 nucleotides in length, 10 to 15nucleotides in length, 10 to 20 nucleotides in length, 15 to 25nucleotides in length, 19 to 21 nucleotides in length, 21 to 23nucleotides in lengths.

In some embodiments, siRNA molecules comprise an antisense strandcomprising a region of complementarity to a target region in a targetmRNA. In some embodiments, the region of complementarity is at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% complementary to a target region in a targetmRNA. In some embodiments, the target region is a region of consecutivenucleotides in the target mRNA. In some embodiments, a complementarynucleotide sequence need not be 100% complementary to that of its targetto be specifically hybridizable or specific for a target RNA sequence.

In some embodiments, siRNA molecules comprise an antisense strand thatcomprises a region of complementarity to a target RNA sequence and theregion of complementarity is in the range of 8 to 15, 8 to 30, 8 to 40,or 10 to 50, or 5 to 50, or 5 to 40 nucleotides in length. In someembodiments, a region of complementarity is 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, or 50 nucleotides in length. In some embodiments, the region ofcomplementarity is complementary with at least 6, at least 7, at least8, at least 9, at least 10, at least 11, at least 12, at least 13, atleast 14, at least 15, at least 16, at least 17, at least 18, at least19, at least 20, at least 21, at least 22, at least 23, at least 24, atleast 25 or more consecutive nucleotides of a target RNA sequence. Insome embodiments, siRNA molecules comprise a nucleotide sequence thatcontains no more than 1, 2, 3, 4, or 5 base mismatches compared to theportion of the consecutive nucleotides of target RNA sequence. In someembodiments, siRNA molecules comprise a nucleotide sequence that has upto 3 mismatches over 15 bases, or up to 2 mismatches over 10 bases.

In some embodiments, siRNA molecules comprise an antisense strandcomprising a nucleotide sequence that is complementary (e.g., at least85%, at least 90%, at least 95%, or 100%) to the target RNA sequence ofthe oligonucleotides provided herein. In some embodiments, siRNAmolecules comprise an antisense strand comprising a nucleotide sequencethat is at least 85%, at least 90%, at least 95%, or 100% identical tothe oligonucleotides provided herein. In some embodiments, siRNAmolecules comprise an antisense strand comprising at least 6, at least7, at least 8, at least 9, at least 10, at least 11, at least 12, atleast 13, at least 14, at least 15, at least 16, at least 17, at least18, at least 19, at least 20, at least 21, at least 22, at least 23, atleast 24, at least 25 or more consecutive nucleotides of theoligonucleotides provided herein.

Double-stranded siRNA may comprise sense and anti-sense RNA strands thatare the same length or different lengths. Double-stranded siRNAmolecules can also be assembled from a single oligonucleotide in astem-loop structure, wherein self-complementary sense and antisenseregions of the siRNA molecule are linked by means of a nucleic acidbased or non-nucleic acid-based linker(s), as well as circularsingle-stranded RNA having two or more loop structures and a stemcomprising self-complementary sense and antisense strands, wherein thecircular RNA can be processed either in vivo or in vitro to generate anactive siRNA molecule capable of mediating RNAi. Small hairpin RNA(shRNA) molecules thus are also contemplated herein. These moleculescomprise a specific antisense sequence in addition to the reversecomplement (sense) sequence, typically separated by a spacer or loopsequence. Cleavage of the spacer or loop provides a single-stranded RNAmolecule and its reverse complement, such that they may anneal to form adsRNA molecule (optionally with additional processing steps that mayresult in addition or removal of one, two, three or more nucleotidesfrom the 3′ end and/or (e.g., and) the 5′ end of either or bothstrands). A spacer can be of a sufficient length to permit the antisenseand sense sequences to anneal and form a double-stranded structure (orstem) prior to cleavage of the spacer (and, optionally, subsequentprocessing steps that may result in addition or removal of one, two,three, four, or more nucleotides from the 3′ end and/or (e.g., and) the5′ end of either or both strands). A spacer sequence is may be anunrelated nucleotide sequence that is situated between two complementarynucleotide sequence regions which, when annealed into a double-strandednucleic acid, comprise a shRNA.

The overall length of the siRNA molecules can vary from about 14 toabout 100 nucleotides depending on the type of siRNA molecule beingdesigned. Generally between about 14 and about 50 of these nucleotidesare complementary to the RNA target sequence, i.e. constitute thespecific antisense sequence of the siRNA molecule. For example, when thesiRNA is a double- or single-stranded siRNA, the length can vary fromabout 14 to about 50 nucleotides, whereas when the siRNA is a shRNA orcircular molecule, the length can vary from about 40 nucleotides toabout 100 nucleotides.

An siRNA molecule may comprise a 3′ overhang at one end of the molecule.The other end may be blunt-ended or have also an overhang (5′ or 3′).When the siRNA molecule comprises an overhang at both ends of themolecule, the length of the overhangs may be the same or different. Inone embodiment, the siRNA molecule of the present disclosure comprises3′ overhangs of about 1 to about 3 nucleotides on both ends of themolecule. In some embodiments, the siRNA molecule comprises 3′ overhangsof about 1 to about 3 nucleotides on the sense strand. In someembodiments, the siRNA molecule comprises 3′ overhangs of about 1 toabout 3 nucleotides on the antisense strand. In some embodiments, thesiRNA molecule comprises 3′ overhangs of about 1 to about 3 nucleotideson both the sense strand and the antisense strand.

In some embodiments, the siRNA molecule comprises one or more modifiednucleotides (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). In someembodiments, the siRNA molecule comprises one or more modifiednucleotides and/or (e.g., and) one or more modified internucleotidelinkages. In some embodiments, the modified nucleotide is a modifiedsugar moiety (e.g. a 2′ modified nucleotide). In some embodiments, thesiRNA molecule comprises one or more 2′ modified nucleotides, e.g., a2′-deoxy, 2′-fluoro (2′-F), 2′-O-methyl (2′-O-Me), 2′-O-methoxyethyl(2′-MOE), 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido(2′-O—NMA). In some embodiments, each nucleotide of the siRNA moleculeis a modified nucleotide (e.g., a 2′-modified nucleotide). In someembodiments, the siRNA molecule comprises one or more phosphorodiamidatemorpholinos. In some embodiments, each nucleotide of the siRNA moleculeis a phosphorodiamidate morpholino.

In some embodiments, the siRNA molecule contains a phosphorothioate orother modified internucleotide linkage. In some embodiments, the siRNAmolecule comprises phosphorothioate internucleoside linkages. In someembodiments, the siRNA molecule comprises phosphorothioateinternucleoside linkages between at least two nucleotides. In someembodiments, the siRNA molecule comprises phosphorothioateinternucleoside linkages between all nucleotides. For example, in someembodiments, the siRNA molecule comprises modified internucleotidelinkages at the first, second, and/or (e.g., and) third internucleosidelinkage at the 5′ or 3′ end of the siRNA molecule.

In some embodiments, the modified internucleotide linkages arephosphorus-containing linkages. In some embodiments,phosphorus-containing linkages that may be used include, but are notlimited to, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates comprising 3′alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates comprising3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′; see U.S. Pat. Nos. 3,687,808; 4,469,863;4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019;5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111;5,563, 253; 5,571,799; 5,587,361; and 5,625,050.

Any of the modified chemistries or formats of siRNA molecules describedherein can be combined with each other. For example, one, two, three,four, five, or more different types of modifications can be includedwithin the same siRNA molecule.

In some embodiments, the antisense strand comprises one or more modifiednucleotides (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). In someembodiments, the antisense strand comprises one or more modifiednucleotides and/or (e.g., and) one or more modified internucleotidelinkages. In some embodiments, the modified nucleotide comprises amodified sugar moiety (e.g. a 2′ modified nucleotide). In someembodiments, the antisense strand comprises one or more 2′ modifiednucleotides, e.g., a 2′-deoxy, 2′-fluoro (2′-F), 2′-O-methyl (2′-O-Me),2′-O-methoxyethyl (2′-MOE), 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or2′-O—N-methylacetamido (2′-O—NMA). In some embodiments, each nucleotideof the antisense strand is a modified nucleotide (e.g., a 2′-modifiednucleotide). In some embodiments, the antisense strand comprises one ormore phosphorodiamidate morpholinos. In some embodiments, the antisensestrand is a phosphorodiamidate morpholino oligomer (PMO).

In some embodiments, antisense strand contains a phosphorothioate orother modified internucleotide linkage. In some embodiments, theantisense strand comprises phosphorothioate internucleoside linkages. Insome embodiments, the antisense strand comprises phosphorothioateinternucleoside linkages between at least two nucleotides. In someembodiments, the antisense strand comprises phosphorothioateinternucleoside linkages between all nucleotides. For example, in someembodiments, the antisense strand comprises modified internucleotidelinkages at the first, second, and/or (e.g., and) third internucleosidelinkage at the 5′ or 3′ end of the siRNA molecule. In some embodiments,the modified internucleotide linkages are phosphorus-containinglinkages. In some embodiments, phosphorus-containing linkages that maybe used include, but are not limited to, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatescomprising 3′alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates comprising 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′; see U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455, 233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563, 253; 5,571,799;5,587,361; and 5,625,050.

Any of the modified chemistries or formats of the antisense stranddescribed herein can be combined with each other. For example, one, two,three, four, five, or more different types of modifications can beincluded within the same antisense strand.

In some embodiments, the sense strand comprises one or more modifiednucleotides (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). In someembodiments, the sense strand comprises one or more modified nucleotidesand/or (e.g., and) one or more modified internucleotide linkages. Insome embodiments, the modified nucleotide is a modified sugar moiety(e.g. a 2′ modified nucleotide). In some embodiments, the sense strandcomprises one or more 2′ modified nucleotides, e.g., a 2′-deoxy,2′-fluoro (2′-F), 2′-O-methyl (2′-O-Me), 2′-O-methoxyethyl (2′-MOE),2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O—NMA). In someembodiments, each nucleotide of the sense strand is a modifiednucleotide (e.g., a 2′-modified nucleotide). In some embodiments, thesense strand comprises one or more phosphorodiamidate morpholinos. Insome embodiments, the antisense strand is a phosphorodiamidatemorpholino oligomer (PMO). In some embodiments, the sense strandcontains a phosphorothioate or other modified internucleotide linkage.In some embodiments, the sense strand comprises phosphorothioateinternucleoside linkages. In some embodiments, the sense strandcomprises phosphorothioate internucleoside linkages between at least twonucleotides. In some embodiments, the sense strand comprisesphosphorothioate internucleoside linkages between all nucleotides. Forexample, in some embodiments, the sense strand comprises modifiedinternucleotide linkages at the first, second, and/or (e.g., and) thirdinternucleoside linkage at the 5′ or 3′ end of the sense strand.

In some embodiments, the modified internucleotide linkages arephosphorus-containing linkages. In some embodiments,phosphorus-containing linkages that may be used include, but are notlimited to, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates comprising 3′alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates comprising3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′; see U.S. Pat. Nos. 3,687,808; 4,469,863;4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019;5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111;5,563, 253; 5,571,799; 5,587,361; and 5,625,050.

Any of the modified chemistries or formats of the sense strand describedherein can be combined with each other. For example, one, two, three,four, five, or more different types of modifications can be includedwithin the same sense strand.

In some embodiments, the antisense or sense strand of the siRNA moleculecomprises modifications that enhance or reduce RNA-induced silencingcomplex (RISC) loading. In some embodiments, the antisense strand of thesiRNA molecule comprises modifications that enhance RISC loading. Insome embodiments, the sense strand of the siRNA molecule comprisesmodifications that reduce RISC loading and reduce off-target effects. Insome embodiments, the antisense strand of the siRNA molecule comprises a2′-O-methoxyethyl (2′-MOE) modification. The addition of the2′-O-methoxyethyl (2′-MOE) group at the cleavage site improves both thespecificity and silencing activity of siRNAs by facilitating theoriented RNA-induced silencing complex (RISC) loading of the modifiedstrand, as described in Song et al., (2017) Mol Ther Nucleic Acids9:242-250, incorporated herein by reference in its entirety. In someembodiments, the antisense strand of the siRNA molecule comprises a2′-OMe-phosphorodithioate modification, which increases RISC loading asdescribed in Wu et al., (2014) Nat Commun 5:3459, incorporated herein byreference in its entirety.

In some embodiments, the sense strand of the siRNA molecule comprises a5′-morpholino, which reduces RISC loading of the sense strand andimproves antisense strand selection and RNAi activity, as described inKumar et al., (2019) Chem Commun (Camb) 55(35):5139-5142, incorporatedherein by reference in its entirety. In some embodiments, the sensestrand of the siRNA molecule is modified with a synthetic RNA-like highaffinity nucleotide analogue, Locked Nucleic Acid (LNA), which reducesRISC loading of the sense strand and further enhances antisense strandincorporation into RISC, as described in Elman et al., (2005) NucleicAcids Res. 33(1): 439-447, incorporated herein by reference in itsentirety. In some embodiments, the sense strand of the siRNA moleculecomprises a 5′ unlocked nucleic acid (UNA) modification, which reduceRISC loading of the sense strand and improve silencing potency of theantisense strand, as described in Snead et al., (2013) Mol Ther NucleicAcids 2(7):e103, incorporated herein by reference in its entirety. Insome embodiments, the sense strand of the siRNA molecule comprises a5-nitroindole modification, which decreased the RNAi potency of thesense strand and reduces off-target effects as described in Zhang etal., (2012) Chembiochem 13(13):1940-1945, incorporated herein byreference in its entirety. In some embodiments, the sense strandcomprises a 2′-O′methyl (2′-O-Me) modification, which reduces RISCloading and the off-target effects of the sense strand, as described inZheng et al., FASEB (2013) 27(10): 4017-4026, incorporated herein byreference in its entirety. In some embodiments, the sense strand of thesiRNA molecule is fully substituted with morpholino, 2′-MOE or 2′-O-Meresidues, and are not recognized by RISC as described in Kole et al.,(2012) Nature reviews. Drug Discovery 11(2):125-140, incorporated hereinby reference in its entirety. In some embodiments the antisense strandof the siRNA molecule comprises a 2′-MOE modification and the sensestrand comprises an 2′-O-Me modification (see e.g., Song et al., (2017)Mol Ther Nucleic Acids 9:242-250). In some embodiments at least one(e.g., at least 2, at least 3, at least 4, at least 5, at least 10)siRNA molecule is linked (e.g., covalently) to a muscle-targeting agent.In some embodiments, the muscle-targeting agent may comprise, or consistof, a nucleic acid (e.g., DNA or RNA), a peptide (e.g., an antibody), alipid (e.g., a microvesicle), or a sugar moiety (e.g., apolysaccharide). In some embodiments, the muscle-targeting agent is anantibody. In some embodiments, the muscle-targeting agent is ananti-transferrin receptor antibody (e.g., any one of the anti-TfRantibodies provided herein). In some embodiments, the muscle-targetingagent may be linked to the 5′ end of the sense strand of the siRNAmolecule. In some embodiments, the muscle-targeting agent may be linkedto the 3′ end of the sense strand of the siRNA molecule. In someembodiments, the muscle-targeting agent may be linked internally to thesense strand of the siRNA molecule. In some embodiments, themuscle-targeting agent may be linked to the 5′ end of the antisensestrand of the siRNA molecule. In some embodiments, the muscle-targetingagent may be linked to the 3′ end of the antisense strand of the siRNAmolecule. In some embodiments, the muscle-targeting agent may be linkedinternally to the antisense strand of the siRNA molecule.

k. microRNA (miRNAs)

In some embodiments, an oligonucleotide may be a microRNA (miRNA).MicroRNAs (referred to as “miRNAs”) are small non-coding RNAs, belongingto a class of regulatory molecules that control gene expression bybinding to complementary sites on a target RNA transcript. Typically,miRNAs are generated from large RNA precursors (termed pri-miRNAs) thatare processed in the nucleus into approximately 70 nucleotidepre-miRNAs, which fold into imperfect stem-loop structures. Thesepre-miRNAs typically undergo an additional processing step within thecytoplasm where mature miRNAs of 18-25 nucleotides in length are excisedfrom one side of the pre-miRNA hairpin by an RNase III enzyme, Dicer.

As used herein, miRNAs including pri-miRNA, pre-miRNA, mature miRNA orfragments of variants thereof that retain the biological activity ofmature miRNA. In one embodiment, the size range of the miRNA can be from21 nucleotides to 170 nucleotides. In one embodiment the size range ofthe miRNA is from 70 to 170 nucleotides in length. In anotherembodiment, mature miRNAs of from 21 to 25 nucleotides in length can beused.

l. Aptamers

In some embodiments, oligonucleotides provided herein may be in the formof aptamers. Generally, in the context of molecular payloads, aptamer isany nucleic acid that binds specifically to a target, such as a smallmolecule, protein, nucleic acid in a cell. In some embodiments, theaptamer is a DNA aptamer or an RNA aptamer. In some embodiments, anucleic acid aptamer is a single-stranded DNA or RNA (ssDNA or ssRNA).It is to be understood that a single-stranded nucleic acid aptamer mayform helices and/or (e.g., and) loop structures. The nucleic acid thatforms the nucleic acid aptamer may comprise naturally occurringnucleotides, modified nucleotides, naturally occurring nucleotides withhydrocarbon linkers (e.g., an alkylene) or a polyether linker (e.g., aPEG linker) inserted between one or more nucleotides, modifiednucleotides with hydrocarbon or PEG linkers inserted between one or morenucleotides, or a combination of thereof. Exemplary publications andpatents describing aptamers and method of producing aptamers include,e.g., Lorsch and Szostak, 1996; Jayasena, 1999; U.S. Pat. Nos.5,270,163; 5,567,588; 5,650,275; 5,670,637; 5,683,867; 5,696,249;5,789,157; 5,843,653; 5,864,026; 5,989,823; 6,569,630; 8,318,438 and PCTapplication WO 99/31275, each incorporated herein by reference.

m. Ribozymes

In some embodiments, oligonucleotides provided herein may be in the formof a ribozyme. A ribozyme (ribonucleic acid enzyme) is a molecule,typically an RNA molecule, that is capable of performing specificbiochemical reactions, similar to the action of protein enzymes.Ribozymes are molecules with catalytic activities including the abilityto cleave at specific phosphodiester linkages in RNA molecules to whichthey have hybridized, such as mRNAs, RNA-containing substrates, lncRNAs,and ribozymes, themselves.

Ribozymes may assume one of several physical structures, one of which iscalled a “hammerhead.” A hammerhead ribozyme is composed of a catalyticcore containing nine conserved bases, a double-stranded stem and loopstructure (stem-loop II), and two regions complementary to the targetRNA flanking regions the catalytic core. The flanking regions enable theribozyme to bind to the target RNA specifically by formingdouble-stranded stems I and III. Cleavage occurs in cis (i.e., cleavageof the same RNA molecule that contains the hammerhead motif) or in trans(cleavage of an RNA substrate other than that containing the ribozyme)next to a specific ribonucleotide triplet by a transesterificationreaction from a 3′, 5′-phosphate diester to a 2′, 3′-cyclic phosphatediester. Without wishing to be bound by theory, it is believed that thiscatalytic activity requires the presence of specific, highly conservedsequences in the catalytic region of the ribozyme.

Modifications in ribozyme structure have also included the substitutionor replacement of various non-core portions of the molecule withnon-nucleotidic molecules. For example, Benseler et al. (J. Am. Chem.Soc. (1993) 115:8483-8484) disclosed hammerhead-like molecules in whichtwo of the base pairs of stem II, and all four of the nucleotides ofloop II were replaced with non-nucleoside linkers based on hexaethyleneglycol, propanediol, bis(triethylene glycol) phosphate,tris(propanediol)bisphosphate, or bis(propanediol) phosphate. Ma et al.(Biochem. (1993) 32:1751-1758; Nucleic Acids Res. (1993) 21:2585-2589)replaced the six nucleotide loop of the TAR ribozyme hairpin withnon-nucleotidic, ethylene glycol-related linkers. Thomson et al.(Nucleic Acids Res. (1993) 21:5600-5603) replaced loop II with linear,non-nucleotidic linkers of 13, 17, and 19 atoms in length.

Ribozyme oligonucleotides can be prepared using well known methods (see,e.g., PCT Publications WO9118624; WO9413688; WO9201806; and WO 92/07065;and U.S. Pat. Nos. 5,436,143 and 5,650,502) or can be purchased fromcommercial sources (e.g., US Biochemicals) and, if desired, canincorporate nucleotide analogs to increase the resistance of theoligonucleotide to degradation by nucleases in a cell. The ribozyme maybe synthesized in any known manner, e.g., by use of a commerciallyavailable synthesizer produced, e.g., by Applied Biosystems, Inc. orMilligen. The ribozyme may also be produced in recombinant vectors byconventional means. See, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory (Current edition). The ribozyme RNA sequencesmay be synthesized conventionally, for example, by using RNA polymerasessuch as T7 or SP6.

n. Guide Nucleic Acids

In some embodiments, oligonucleotides are guide nucleic acid, e.g.,guide RNA (gRNA) molecules. Generally, a guide RNA is a short syntheticRNA composed of (1) a scaffold sequence that binds to a nucleic acidprogrammable DNA binding protein (napDNAbp), such as Cas9, and (2) anucleotide spacer portion that defines the DNA target sequence (e.g.,genomic DNA target) to which the gRNA binds in order to bring thenucleic acid programmable DNA binding protein in proximity to the DNAtarget sequence. In some embodiments, the napDNAbp is a nucleicacid-programmable protein that forms a complex with (e.g., binds orassociates with) one or more RNA(s) that targets the nucleicacid-programmable protein to a target DNA sequence (e.g., a targetgenomic DNA sequence). In some embodiments, a nucleic acid-programmablenuclease, when in a complex with an RNA, may be referred to as anuclease:RNA complex. Guide RNAs can exist as a complex of two or moreRNAs, or as a single RNA molecule.

Guide RNAs (gRNAs) that exist as a single RNA molecule may be referredto as single-guide RNAs (sgRNAs), though gRNA is also used to refer toguide RNAs that exist as either single molecules or as a complex of twoor more molecules. Typically, gRNAs that exist as a single RNA speciescomprise two domains: (1) a domain that shares homology to a targetnucleic acid (i.e., directs binding of a Cas9 complex to the target);and (2) a domain that binds a Cas9 protein. In some embodiments, domain(2) corresponds to a sequence known as a tracrRNA and comprises astem-loop structure. In some embodiments, domain (2) is identical orhomologous to a tracrRNA as provided in Jinek et al., Science337:816-821 (2012), the entire contents of which is incorporated hereinby reference.

In some embodiments, a gRNA comprises two or more of domains (1) and(2), and may be referred to as an extended gRNA. For example, anextended gRNA will bind two or more Cas9 proteins and bind a targetnucleic acid at two or more distinct regions, as described herein. ThegRNA comprises a nucleotide sequence that complements a target site,which mediates binding of the nuclease/RNA complex to said target site,providing the sequence specificity of the nuclease:RNA complex. In someembodiments, the RNA-programmable nuclease is the (CRISPR-associatedsystem) Cas9 endonuclease, for example, Cas9 (Csn1) from Streptococcuspyogenes (see, e.g., “Complete genome sequence of an M1 strain ofStreptococcus pyogenes.” Ferretti J. J., McShan W. M., Ajdic D. J.,Savic D. J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A. N.,Kenton S., Lai H. S., Lin S. P., Qian Y., Jia H. G., Najar F. Z., RenQ., Zhu H., Song L., White J., Yuan X., Clifton S. W., Roe B. A.,McLaughlin R. E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663 (2001);“CRISPR RNA maturation by trans-encoded small RNA and host factor RNaseIII.” Deltcheva E., Chylinski K., Sharma C. M., Gonzales K., Chao Y.,Pirzada Z. A., Eckert M. R., Vogel J., Charpentier E., Nature471:602-607 (2011); and “A programmable dual-RNA-guided DNA endonucleasein adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I.,Hauer M., Doudna J. A., Charpentier E. Science 337:816-821 (2012), theentire contents of each of which are incorporated herein by reference.

o. Multimers

In some embodiments, molecular payloads may comprise multimers (e.g.,concatemers) of 2 or more oligonucleotides connected by a linker. Inthis way, in some embodiments, the oligonucleotide loading of acomplex/conjugate can be increased beyond the available linking sites ona targeting agent (e.g., available thiol sites on an antibody) orotherwise tuned to achieve a particular payload loading content.Oligonucleotides in a multimer can be the same or different (e.g.,targeting different genes or different sites on the same gene orproducts thereof).

In some embodiments, multimers comprise 2 or more oligonucleotideslinked together by a cleavable linker. However, in some embodiments,multimers comprise 2 or more oligonucleotides linked together by anon-cleavable linker. In some embodiments, a multimer comprises 2, 3, 4,5, 6, 7, 8, 9, 10 or more oligonucleotides linked together. In someembodiments, a multimer comprises 2 to 5, 2 to 10 or 4 to 20oligonucleotides linked together.

In some embodiments, a multimer comprises 2 or more oligonucleotideslinked end-to-end (in a linear arrangement). In some embodiments, amultimer comprises 2 or more oligonucleotides linked end-to-end via anoligonucleotide based linker (e.g., poly-dT linker, an abasic linker).In some embodiments, a multimer comprises a 5′ end of oneoligonucleotide linked to a 3′ end of another oligonucleotide. In someembodiments, a multimer comprises a 3′ end of one oligonucleotide linkedto a 3′ end of another oligonucleotide. In some embodiments, a multimercomprises a 5′ end of one oligonucleotide linked to a 5′ end of anotheroligonucleotide. Still, in some embodiments, multimers can comprise abranched structure comprising multiple oligonucleotides linked togetherby a branching linker.

Further examples of multimers that may be used in the complexes providedherein are disclosed, for example, in US Patent Application Number2015/0315588 A1, entitled Methods of delivering multiple targetingoligonucleotides to a cell using cleavable linkers, which was publishedon Nov. 5, 2015; US Patent Application Number 2015/0247141 A1, entitledMultimeric Oligonucleotide Compounds, which was published on Sep. 3,2015, US Patent Application Number US 2011/0158937 A1, entitledImmunostimulatory Oligonucleotide Multimers, which was published on Jun.30, 2011; and U.S. Pat. No. 5,693,773, entitled Triplex-FormingAntisense Oligonucleotides Having Abasic Linkers Targeting Nucleic AcidsComprising Mixed Sequences Of Purines And Pyrimidines, which issued onDec. 2, 1997, the contents of each of which are incorporated herein byreference in their entireties.

ii. Small Molecules:

Any suitable small molecule may be used as a molecular payload, asdescribed herein. In some embodiments, the small molecule is asdescribed in US Patent Application Publication 2016052914A1, publishedon Feb. 25, 2016, entitled “Compounds And Methods For Myotonic DystrophyTherapy”. Further examples of small molecule payloads are provided inLopez-Morato M, et al., Small Molecules Which Improve Pathogenesis ofMyotonic Dystrophy Type 1, (Review) Front. Neurol., 18 May 2018. Forexample, in some embodiments, the small molecule is an MBNL1 upregulatorsuch as phenylbuthazone, ketoprofen, ISOX, or vorinostat. In someembodiments, the small molecule is an H-Ras pathway inhibitor such asmanumycin A. In some embodiments, the small molecule is a protein kinasemodulator such as Ro-318220, C16, C51, Metformin, AICAR, lithiumchloride, TDZD-8 or Bio. In some embodiments, the small molecule is aplant alkaloid such as harmine. In some embodiments, the small moleculeis a transcription inhibitor such as pentamidine, propamidine,heptamidiine or actinomycin D. In some embodiments, the small moleculeis an inhibitor of Glycogen synthase kinase 3 beta (GSK3B), for example,as disclosed in Jones K, et al., GSK3(3 mediates muscle pathology inmyotonic dystrophy. J Clin Invest. 2012 December; 122(12):4461-72; andWei C, et al., GSK3β is a new therapeutic target for myotonic dystrophytype 1. Rare Dis. 2013; 1: e26555; and Palomo V, et al., SubtlyModulating Glycogen Synthase Kinase 3 β: Allosteric InhibitorDevelopment and Their Potential for the Treatment of Chronic Diseases. JMed Chem. 2017 Jun. 22; 60(12):4983-5001, the contents of each of whichare incorporated herein by reference in their entireties. In someembodiments, the small molecule is a substitutedpyrido[2,3-d]pyrimidines and pentamidine-like compound, as disclosed inGonzalez Ala., et al., In silico discovery of substitutedpyrido[2,3-d]pyrimidines and pentamidine-like compounds with biologicalactivity in myotonic dystrophy models. PLoS One. 2017 Jun. 5;12(6):e0178931, the contents of which are incorporated herein byreference in its entirety. In some embodiments, the small molecule is anMBNL1 modulator, for example, as disclosed in: Zhange F, et al., A flowcytometry-based screen identifies MBNL1 modulators that rescue splicingdefects in myotonic dystrophy type I. Hum Mol Genet. 2017 Aug. 15;26(16):3056-3068, the contents of which are incorporated herein byreference in its entirety.

iii. Peptides

Any suitable peptide or protein may be used as a molecular payload, asdescribed herein. A peptide or protein payload may correspond to asequence of a protein that preferentially binds to a nucleic acid, e.g.a disease-associated repeat, or a protein, e.g. MBNL1, found in musclecells. In some embodiments, peptides or proteins may be produced,synthesized, and/or (e.g., and) derivatized using several methodologies,e.g. phage displayed peptide libraries, one-bead one-compound peptidelibraries, or positional scanning synthetic peptide combinatoriallibraries. Exemplary methodologies have been characterized in the artand are incorporated by reference (Gray, B. P. and Brown, K. C.“Combinatorial Peptide Libraries: Mining for Cell-Binding Peptides” ChemRev. 2014, 114:2, 1020-1081; Samoylova, T. I. and Smith, B. F.“Elucidation of muscle-binding peptides by phage display screening.”Muscle Nerve, 1999, 22:4. 460-6).

In some embodiments, the peptide is as described in US PatentApplication 2018/0021449, published on Jan. 25, 2018, “Antisenseconjugates for decreasing expression of DMPK”. In some embodiments, thepeptide is as described in Garcia-Lopez et al., “In vivo discovery of apeptide that prevents CUG—RNA hairpin formation and reverses RNAtoxicity in myotonic dystrophy models”, PNAS Jul. 19, 2011. 108 (29)11866-11871. In some embodiments, the peptide or protein may target,e.g., bind to, a disease-associated repeat, e.g. an RNA CUG repeatexpansion.

In some embodiments, the peptide or protein comprises a fragment of anMBNL protein, e.g., MBNL1. In some embodiments, the peptide or proteincomprises at least one zinc finger. In some embodiments, the peptide orprotein may comprise about 2-25 amino acids, about 2-20 amino acids,about 2-15 amino acids, about 2-10 amino acids, or about 2-5 aminoacids. The peptide or protein may comprise naturally-occurring aminoacids, e.g. cysteine, alanine, or non-naturally-occurring or modifiedamino acids. Non-naturally occurring amino acids include β-amino acids,homo-amino acids, proline derivatives, 3-substituted alaninederivatives, linear core amino acids, N-methyl amino acids, and othersknown in the art. In some embodiments, the peptide may be linear; inother embodiments, the peptide may be cyclic, e.g. bicyclic.

iv. Nucleic Acid Constructs

Any suitable gene expression construct may be used as a molecularpayload, as described herein. In some embodiments, a gene expressionconstruct may be a vector or a cDNA fragment. In some embodiments, agene expression construct may be messenger RNA (mRNA). In someembodiments, a mRNA used herein may be a modified mRNA, e.g., asdescribed in U.S. Pat. No. 8,710,200, issued on Apr. 24, 2014, entitled“Engineered nucleic acids encoding a modified erythropoietin and theirexpression”. In some embodiments, a mRNA may comprise a 5′-methyl cap.In some embodiments, a mRNA may comprise a polyA tail, optionally of upto 160 nucleotides in length. A gene expression construct may encode asequence of a protein that preferentially binds to a nucleic acid, e.g.a disease-associated repeat, or a protein, e.g. MBNL1, found in musclecells. In some embodiments, the gene expression construct may beexpressed, e.g., overexpressed, within the nucleus of a muscle cell. Insome embodiments, the gene expression construct encodes a MBNL protein,e.g., MBNL1. In some embodiments, the gene expression construct encodesa protein that comprises at least one zinc finger. In some embodiments,the gene expression construct encodes a protein that binds to adisease-associated repeat. In some embodiments, the gene expressionconstruct encodes a protein that leads to a reduction in the expressionof a disease-associated repeat. In some embodiments, the gene expressionconstruct encodes a gene editing enzyme. Additional examples of nucleicacid constructs that may be used as molecular payloads are provided inInternational Patent Application Publication WO2017152149A1, publishedon Sep. 19, 2017, entitled, “Closed-Ended Linear Duplex Dna ForNon-Viral Gene Transfer”; U.S. Pat. No. 8,853,377B2, issued on Oct. 7,2014, entitled, “mRNA For Use In Treatment Of Human Genetic Diseases”;and U.S. Pat. No. 8,822,663B2, issued on Sep. 2, 2014, EngineeredNucleic Acids And Methods Of Use Thereof,” the contents of each of whichare incorporated herein by reference in their entireties.

C. Linkers

Complexes described herein generally comprise a linker that connects anyone of the anti-TfR antibodies described herein to a molecular payload.A linker comprises at least one covalent bond. In some embodiments, alinker may be a single bond, e.g., a disulfide bond or disulfide bridge,that connects an anti-TfR antibody to a molecular payload. However, insome embodiments, a linker may connect any one of the anti-TfRantibodies described herein to a molecular payload through multiplecovalent bonds. In some embodiments, a linker may be a cleavable linker.However, in some embodiments, a linker may be a non-cleavable linker. Alinker is generally stable in vitro and in vivo, and may be stable incertain cellular environments. Additionally, generally a linker does notnegatively impact the functional properties of either the anti-TfRantibody or the molecular payload. Examples and methods of synthesis oflinkers are known in the art (see, e.g. Kline, T. et al. “Methods toMake Homogenous Antibody Drug Conjugates.” Pharmaceutical Research,2015, 32:11, 3480-3493; Jain, N. et al. “Current ADC Linker Chemistry”Pharm Res. 2015, 32:11, 3526-3540; McCombs, J. R. and Owen, S. C.“Antibody Drug Conjugates: Design and Selection of Linker, Payload andConjugation Chemistry” AAPS J. 2015, 17:2, 339-351).

A precursor to a linker typically will contain two different reactivespecies that allow for attachment to both the anti-TfR antibody and amolecular payload. In some embodiments, the two different reactivespecies may be a nucleophile and/or (e.g., and) an electrophile. In someembodiments, a linker is connected to an anti-TfR antibody viaconjugation to a lysine residue or a cysteine residue of the anti-TfRantibody. In some embodiments, a linker is connected to a cysteineresidue of an anti-TfR antibody via a maleimide-containing linker,wherein optionally the maleimide-containing linker comprises amaleimidocaproyl or maleimidomethyl cyclohexane-1-carboxylate group. Insome embodiments, a linker is connected to a cysteine residue of ananti-TfR antibody or thiol functionalized molecular payload via a3-arylpropionitrile functional group. In some embodiments, a linker isconnected to a lysine residue of an anti-TfR antibody. In someembodiments, a linker is connected to an anti-TfR antibody and/or (e.g.,and) a molecular payload via an amide bond, a carbamate bond, ahydrazide, a trizaole, a thioether, or a disulfide bond.

i. Cleavable Linkers

A cleavable linker may be a protease-sensitive linker, a pH-sensitivelinker, or a glutathione-sensitive linker. These linkers are generallycleavable only intracellularly and are preferably stable inextracellular environments, e.g. extracellular to a muscle cell.

Protease-sensitive linkers are cleavable by protease enzymatic activity.These linkers typically comprise peptide sequences and may be 2-10 aminoacids, about 2-5 amino acids, about 5-10 amino acids, about 10 aminoacids, about 5 amino acids, about 3 amino acids, or about 2 amino acidsin length. In some embodiments, a peptide sequence may comprisenaturally-occurring amino acids, e.g. cysteine, alanine, ornon-naturally-occurring or modified amino acids. Non-naturally occurringamino acids include β-amino acids, homo-amino acids, prolinederivatives, 3-substituted alanine derivatives, linear core amino acids,N-methyl amino acids, and others known in the art. In some embodiments,a protease-sensitive linker comprises a valine-citrulline oralanine-citrulline dipeptide sequence. In some embodiments, aprotease-sensitive linker can be cleaved by a lysosomal protease, e.g.cathepsin B, and/or (e.g., and) an endosomal protease.

A pH-sensitive linker is a covalent linkage that readily degrades inhigh or low pH environments. In some embodiments, a pH-sensitive linkermay be cleaved at a pH in a range of 4 to 6. In some embodiments, apH-sensitive linker comprises a hydrazone or cyclic acetal. In someembodiments, a pH-sensitive linker is cleaved within an endosome or alysosome.

In some embodiments, a glutathione-sensitive linker comprises adisulfide moiety. In some embodiments, a glutathione-sensitive linker iscleaved by a disulfide exchange reaction with a glutathione speciesinside a cell. In some embodiments, the disulfide moiety furthercomprises at least one amino acid, e.g. a cysteine residue.

In some embodiments, the linker is a Val-cit linker (e.g., as describedin U.S. Pat. No. 6,214,345, incorporated herein by reference). In someembodiments, before conjugation, the val-cit linker has a structure of:

In some embodiments, after conjugation, the val-cit linker has astructure of:

In some embodiments, the Val-cit linker is attached to a reactivechemical moiety (e.g., SPAAC for click chemistry conjugation). In someembodiments, before click chemistry conjugation, the val-cit linkerattached to a reactive chemical moiety (e.g., SPAAC for click chemistryconjugation) has the structure of:

wherein n is any number from 0-10. In some embodiments, n is 3.

In some embodiments, the val-cit linker attached to a reactive chemicalmoiety (e.g., SPAAC for click chemistry conjugation) is conjugated(e.g., via a different chemical moiety) to a molecular payload (e.g., anoligonucleotide). In some embodiments, the val-cit linker attached to areactive chemical moiety (e.g., SPAAC for click chemistry conjugation)and conjugated to a molecular payload (e.g., an oligonucleotide) has thestructure of (before click chemistry conjugation):

wherein n is any number from 0-10. In some embodiments, n is 3.

In some embodiments, after conjugation to a molecular payload (e.g., anoligonucleotide), the val-cit linker has a structure of:

wherein n is any number from 0-10, and wherein m is any number from0-10. In some embodiments, n is 3 and m is 4.

ii. Non-Cleavable Linkers

In some embodiments, non-cleavable linkers may be used. Generally, anon-cleavable linker cannot be readily degraded in a cellular orphysiological environment. In some embodiments, a non-cleavable linkercomprises an optionally substituted alkyl group, wherein thesubstitutions may include halogens, hydroxyl groups, oxygen species, andother common substitutions. In some embodiments, a linker may comprisean optionally substituted alkyl, an optionally substituted alkylene, anoptionally substituted arylene, a heteroarylene, a peptide sequencecomprising at least one non-natural amino acid, a truncated glycan, asugar or sugars that cannot be enzymatically degraded, an azide, analkyne-azide, a peptide sequence comprising a LPXT sequence, athioether, a biotin, a biphenyl, repeating units of polyethylene glycolor equivalent compounds, acid esters, acid amides, sulfamides, and/or(e.g., and) an alkoxy-amine linker. In some embodiments,sortase-mediated ligation will be utilized to covalently link ananti-TfR antibody comprising a LPXT sequence to a molecular payloadcomprising a (G)_(n) sequence (see, e.g. Proft T. Sortase-mediatedprotein ligation: an emerging biotechnology tool for proteinmodification and immobilization. Biotechnol Lett. 2010, 32(1):1-10).

In some embodiments, a linker may comprise a substituted alkylene, anoptionally substituted alkenylene, an optionally substituted alkynylene,an optionally substituted cycloalkylene, an optionally substitutedcycloalkenylene, an optionally substituted arylene, an optionallysubstituted heteroarylene further comprising at least one heteroatomselected from N, O, and S; an optionally substituted heterocyclylenefurther comprising at least one heteroatom selected from N, O, and S; animino, an optionally substituted nitrogen species, an optionallysubstituted oxygen species O, an optionally substituted sulfur species,or a poly(alkylene oxide), e.g. polyethylene oxide or polypropyleneoxide.

iii. Linker conjugation

In some embodiments, a linker is connected to an anti-TfR antibodyand/or (e.g., and) molecular payload via a phosphate, thioether, ether,carbon-carbon, carbamate, or amide bond. In some embodiments, a linkeris connected to an oligonucleotide through a phosphate orphosphorothioate group, e.g. a terminal phosphate of an oligonucleotidebackbone. In some embodiments, a linker is connected to an anti-TfRantibody, through a lysine or cysteine residue present on the anti-TfRantibody.

In some embodiments, a linker is connected to an anti-TfR antibodyand/or (e.g., and) molecular payload by a cycloaddition reaction betweenan azide and an alkyne to form a triazole, wherein the azide and thealkyne may be located on the anti-TfR antibody, molecular payload, orthe linker. In some embodiments, an alkyne may be a cyclic alkyne, e.g.,a cyclooctyne. In some embodiments, an alkyne may be bicyclononyne (alsoknown as bicyclo[6.1.0]nonyne or BCN) or substituted bicyclononyne. Insome embodiments, a cyclooctane is as described in International PatentApplication Publication WO2011136645, published on Nov. 3, 2011,entitled, “Fused Cyclooctyne Compounds And Their Use In Metal free ClickReactions”. In some embodiments, an azide may be a sugar or carbohydratemolecule that comprises an azide. In some embodiments, an azide may be6-azido-6-deoxygalactose or 6-azido-N-acetylgalactosamine. In someembodiments, a sugar or carbohydrate molecule that comprises an azide isas described in International Patent Application PublicationWO2016170186, published on Oct. 27, 2016, entitled, “Process For TheModification Of A Glycoprotein Using A Glycosyltransferase That Is Or IsDerived From A β(1,4)-N-Acetylgalactosaminyltransferase”. In someembodiments, a cycloaddition reaction between an azide and an alkyne toform a triazole, wherein the azide and the alkyne may be located on theanti-TfR antibody, molecular payload, or the linker is as described inInternational Patent Application Publication WO2014065661, published onMay 1, 2014, entitled, “Modified antibody, antibody-conjugate andprocess for the preparation thereof”; or International PatentApplication Publication WO2016170186, published on Oct. 27, 2016,entitled, “Process For The Modification Of A Glycoprotein Using AGlycosyltransferase That Is Or Is Derived From Aβ(1,4)-N-Acetylgalactosaminyltransferase”.

In some embodiments, a linker further comprises a spacer, e.g., apolyethylene glycol spacer or an acyl/carbomoyl sulfamide spacer, e.g.,a HydraSpace™ spacer. In some embodiments, a spacer is as described inVerkade, J. M. M. et al., “A Polar Sulfamide Spacer SignificantlyEnhances the Manufacturability, Stability, and Therapeutic Index ofAntibody-Drug Conjugates”, Antibodies, 2018, 7, 12.

In some embodiments, a linker is connected to an anti-TfR antibodyand/or (e.g., and) molecular payload by the Diels-Alder reaction betweena dienophile and a diene/hetero-diene, wherein the dienophile and thediene/hetero-diene may be located on the anti-TfR antibody, molecularpayload, or the linker. In some embodiments a linker is connected to ananti-TfR antibody and/or (e.g., and) molecular payload by otherpericyclic reactions, e.g. ene reaction. In some embodiments, a linkeris connected to an anti-TfR antibody and/or (e.g., and) molecularpayload by an amide, thioamide, or sulfonamide bond reaction. In someembodiments, a linker is connected to an anti-TfR antibody and/or (e.g.,and) molecular payload by a condensation reaction to form an oxime,hydrazone, or semicarbazide group existing between the linker and theanti-TfR antibody and/or (e.g., and) molecular payload.

In some embodiments, a linker is connected to an anti-TfR antibodyand/or (e.g., and) molecular payload by a conjugate addition reactionsbetween a nucleophile, e.g. an amine or a hydroxyl group, and anelectrophile, e.g. a carboxylic acid, carbonate, or an aldehyde. In someembodiments, a nucleophile may exist on a linker and an electrophile mayexist on an anti-TfR antibody or molecular payload prior to a reactionbetween a linker and an anti-TfR antibody or molecular payload. In someembodiments, an electrophile may exist on a linker and a nucleophile mayexist on an anti-TfR antibody or molecular payload prior to a reactionbetween a linker and an anti-TfR antibody or molecular payload. In someembodiments, an electrophile may be an azide, pentafluorophenyl, asilicon centers, a carbonyl, a carboxylic acid, an anhydride, anisocyanate, a thioisocyanate, a succinimidyl ester, a sulfosuccinimidylester, a maleimide, an alkyl halide, an alkyl pseudohalide, an epoxide,an episulfide, an aziridine, an aryl, an activated phosphorus center,and/or (e.g., and) an activated sulfur center. In some embodiments, anucleophile may be an optionally substituted alkene, an optionallysubstituted alkyne, an optionally substituted aryl, an optionallysubstituted heterocyclyl, a hydroxyl group, an amino group, analkylamino group, an anilido group, or a thiol group.

In some embodiments, the val-cit linker attached to a reactive chemicalmoiety (e.g., SPAAC for click chemistry conjugation) is conjugated tothe anti-TfR antibody by a structure of:

wherein m is any number from 0-10. In some embodiments, m is 4.

In some embodiments, the val-cit linker attached to a reactive chemicalmoiety (e.g., SPAAC for click chemistry conjugation) is conjugated to ananti-TfR antibody having a structure of:

wherein m is any number from 0-10. In some embodiments, m is 4.

In some embodiments, the val-cit linker attached to a reactive chemicalmoiety (e.g., SPAAC for click chemistry conjugation) and conjugated toan anti-TfR antibody has a structure of:

wherein n is any number from 0-10, wherein m is any number from 0-10. Insome embodiments, n is 3 and/or (e.g., and) m is 4.

In some embodiments, the val-cit linker that links the antibody and themolecular payload has a structure of:

wherein n is any number from 0-10, wherein m is any number from 0-10. Insome embodiments, n is 3 and/or (e.g., and) m is 4. In some embodiments,n is 3 and/or (e.g., and) m is 4. In some embodiments, X is NH (e.g., NHfrom an amine group of a lysine), S (e.g., S from a thiol group of acysteine), or O (e.g., O from a hydroxyl group of a serine, threonine,or tyrosine) of the antibody.

In some embodiments, the val-cit linker used to covalently link ananti-TfR antibody and a molecular payload (e.g., an oligonucleotide) hasa structure of:

wherein n is any number from 0-10, wherein m is any number from 0-10. Insome embodiments, n is 3 and m is 4.

In structures formula (A), (B), (C), and (D), L1, in some embodiments,is a spacer that is a substituted or unsubstituted aliphatic,substituted or unsubstituted heteroaliphatic, substituted orunsubstituted carbocyclylene, substituted or unsubstitutedheterocyclylene, substituted or unsubstituted arylene, substituted orunsubstituted heteroarylene, —O—, —N(R^(A))—, —S—, —C(═O)—, —C(═O)O—,—C(═O)NR^(A)—, —NR^(A)C(═O)—, —NR^(A)C(═O)R^(A)—, —C(═O)R^(A)—,—NR^(A)C(═O)O—, —NR^(A)C(═O)N(R^(A))—, —OC(═O)—, —OC(═O)O—,—OC(═O)N(R^(A))—, —S(O)₂NR^(A)—, —NR^(A)S(O)₂—, or a combinationthereof. In some embodiments, L1 is

wherein the piperazine moiety links to the oligonucleotide, wherein L2is

In some embodiments, L1 is:

wherein the piperazine moiety links to the oligonucleotide.

In some embodiments, L1 is

In some embodiments, L1 is linked to a 5′ phosphate of theoligonucleotide.

In some embodiments, L1 is optional (e.g., need not be present).

In some embodiments, any one of the complexes described herein has astructure of:

wherein n is 0-15 (e.g., 3) and m is 0-15 (e.g., 4).

C. Examples of Antibody-Molecular Payload Complexes

Further provided herein are non-limiting examples of complexescomprising any one the anti-TfR antibodies described herein covalentlylinked to any of the molecular payloads (e.g., an oligonucleotide)described herein. In some embodiments, the anti-TfR antibody (e.g., anyone of the anti-TfR antibodies provided in Table 2) is covalently linkedto a molecular payload (e.g., an oligonucleotide) via a linker. Any ofthe linkers described herein may be used. In some embodiments, if themolecular payload is an oligonucleotide, the linker is linked to the 5′end, the 3′ end, or internally of the oligonucleotide. In someembodiments, the linker is linked to the anti-TfR antibody via athiol-reactive linkage (e.g., via a cysteine in the anti-TfR antibody).In some embodiments, the linker (e.g., a Val-cit linker) is linked tothe antibody (e.g., an anti-TfR antibody described herein) via an aminegroup (e.g., via a lysine in the antibody). In some embodiments, themolecular payload is a DMPK-targeting oligonucleotide (e.g., aDMPK-targeting oligonucleotide listed in Table 8 or Table 17).

An example of a structure of a complex comprising an anti-TfR antibodycovalently linked to a molecular payload via a Val-cit linker isprovided below:

wherein the linker is linked to the antibody via a thiol-reactivelinkage (e.g., via a cysteine in the antibody). In some embodiments, themolecular payload is a DMPK-targeting oligonucleotide (e.g., aDMPK-targeting oligonucleotide listed in Table 8 or Table 17).

Another example of a structure of a complex comprising an anti-TfRantibody covalently linked to a molecular payload via a Val-cit linkeris provided below:

wherein n is a number between 0-10, wherein m is a number between 0-10,wherein the linker is linked to the antibody via an amine group (e.g.,on a lysine residue), and/or (e.g., and) wherein the linker is linked tothe oligonucleotide (e.g., at the 5′ end, 3′ end, or internally). Insome embodiments, the linker is linked to the antibody via a lysine, thelinker is linked to the oligonucleotide at the 5′ end, n is 3, and m is4. In some embodiments, the molecular payload is an oligonucleotidecomprising a sense strand and an antisense strand, and, the linker islinked to the sense strand or the antisense strand at the 5′ end or the3′ end. In some embodiments, the molecular payload is a DMPK-targetingoligonucleotide (e.g., a DMPK-targeting oligonucleotide listed in Table8 or Table 17).

It should be appreciated that antibodies can be linked to molecularpayloads with different stoichiometries, a property that may be referredto as a drug to antibody ratios (DAR) with the “drug” being themolecular payload. In some embodiments, one molecular payload is linkedto an antibody (DAR=1). In some embodiments, two molecular payloads arelinked to an antibody (DAR=2). In some embodiments, three molecularpayloads are linked to an antibody (DAR=3). In some embodiments, fourmolecular payloads are linked to an antibody (DAR=4). In someembodiments, a mixture of different complexes, each having a differentDAR, is provided. In some embodiments, an average DAR of complexes insuch a mixture may be in a range of 1 to 3, 1 to 4, 1 to 5 or more. DARmay be increased by conjugating molecular payloads to different sites onan antibody and/or (e.g., and) by conjugating multimers to one or moresites on antibody. For example, a DAR of 2 may be achieved byconjugating a single molecular payload to two different sites on anantibody or by conjugating a dimer molecular payload to a single site ofan antibody.

In some embodiments, the complex described herein comprises an anti-TfRantibody described herein (e.g., the 3-A4, 3-M12, and 5-H12 antibodiesprovided in Table 2 in an IgG or Fab form) covalently linked to amolecular payload. In some embodiments, the complex described hereincomprises an anti-TfR antibody described herein (e.g., the 3-A4, 3-M12,and 5-H12 antibodies provided in Table 2 in a IgG or Fab form)covalently linked to molecular payload via a linker (e.g., a Val-citlinker). In some embodiments, the linker (e.g., a Val-cit linker) islinked to the antibody (e.g., an anti-TfR antibody described herein) viaa thiol-reactive linkage (e.g., via a cysteine in the antibody). In someembodiments, the linker (e.g., a Val-cit linker) is linked to theantibody (e.g., an anti-TfR antibody described herein) via an aminegroup (e.g., via a lysine in the antibody). In some embodiments, themolecular payload is a DMPK-targeting oligonucleotide (e.g., aDMPK-targeting oligonucleotide listed in Table 8 or Table 17).

In some embodiments, in any one of the examples of complexes describedherein, the molecular payload a DMPK-targeting oligonucleotide (e.g., aDMPK-targeting oligonucleotide listed in Table 8 or Table 17).

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a CDR-H1, a CDR-H2, and a CDR-H3 that are the same asthe CDR-H1, CDR-H2, and CDR-H3 shown in Table 2; and a CDR-L1, a CDR-L2,and a CDR-L3 that are the same as the CDR-L1, CDR-L2, and CDR-L3 shownin Table 2. In some embodiments, the molecular payload is aDMPK-targeting oligonucleotide (e.g., a DMPK-targeting oligonucleotidelisted in Table 8 or Table 17).

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a VH comprising the amino acid sequence of SEQ ID NO:69, SEQ ID NO: 71, or SEQ ID NO: 72, and a VL comprising the amino acidsequence of SEQ ID NO: 70. In some embodiments, the molecular payload isa DMPK-targeting oligonucleotide (e.g., a DMPK-targeting oligonucleotidelisted in Table 8 or Table 17).

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a VH comprising the amino acid sequence of SEQ ID NO:73 or SEQ ID NO: 76, and a VL comprising the amino acid sequence of SEQID NO: 74. In some embodiments, the molecular payload is aDMPK-targeting oligonucleotide (e.g., a DMPK-targeting oligonucleotidelisted in Table 8 or Table 17).

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a VH comprising the amino acid sequence of SEQ ID NO:73 or SEQ ID NO: 76, and a VL comprising the amino acid sequence of SEQID NO: 75. In some embodiments, the molecular payload is aDMPK-targeting oligonucleotide (e.g., a DMPK-targeting oligonucleotidelisted in Table 8 or Table 17).

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a VH comprising the amino acid sequence of SEQ ID NO:77, and a VL comprising the amino acid sequence of SEQ ID NO: 78. Insome embodiments, the molecular payload is a DMPK-targetingoligonucleotide (e.g., a DMPK-targeting oligonucleotide listed in Table8 or Table 17).

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a VH comprising the amino acid sequence of SEQ ID NO:77 or SEQ ID NO: 79, and a VL comprising the amino acid sequence of SEQID NO: 80. In some embodiments, the molecular payload is aDMPK-targeting oligonucleotide (e.g., a DMPK-targeting oligonucleotidelisted in Table 8 or Table 17).

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 84, SEQ ID NO: 86 or SEQ ID NO: 87 and a light chaincomprising the amino acid sequence of SEQ ID NO: 85. In someembodiments, the molecular payload is a DMPK-targeting oligonucleotide(e.g., a DMPK-targeting oligonucleotide listed in Table 8 or Table 17).

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 88 or SEQ ID NO: 91, and a light chain comprising the aminoacid sequence of SEQ ID NO: 89. In some embodiments, the molecularpayload is a DMPK-targeting oligonucleotide (e.g., a DMPK-targetingoligonucleotide listed in Table 8 or Table 17).

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 88 or SEQ ID NO: 91, and a light chain comprising the aminoacid sequence of SEQ ID NO: 90. In some embodiments, the molecularpayload is a DMPK-targeting oligonucleotide (e.g., a DMPK-targetingoligonucleotide listed in Table 8 or Table 17).

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 92 or SEQ ID NO: 94, and a light chain comprising the aminoacid sequence of SEQ ID NO: 95. In some embodiments, the molecularpayload is a DMPK-targeting oligonucleotide (e.g., a DMPK-targetingoligonucleotide listed in Table 8 or Table 17).

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 92, and a light chain comprising the amino acid sequence ofSEQ ID NO: 93. In some embodiments, the molecular payload is aDMPK-targeting oligonucleotide (e.g., a DMPK-targeting oligonucleotidelisted in Table 8 or Table 17).

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 97, SEQ ID NO: 98, or SEQ ID NO: 99 and a VL comprising theamino acid sequence of SEQ ID NO: 85. In some embodiments, the molecularpayload is a DMPK-targeting oligonucleotide (e.g., a DMPK-targetingoligonucleotide listed in Table 8 or Table 17).

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 100 or SEQ ID NO: 101 and a light chain comprising the aminoacid sequence of SEQ ID NO: 89. In some embodiments, the molecularpayload is a DMPK-targeting oligonucleotide (e.g., a DMPK-targetingoligonucleotide listed in Table 8 or Table 17).

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 100 or SEQ ID NO: 101 and a light chain comprising the aminoacid sequence of SEQ ID NO: 90. In some embodiments, the molecularpayload is a DMPK-targeting oligonucleotide (e.g., a DMPK-targetingoligonucleotide listed in Table 8 or Table 17).

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 102 and a light chain comprising the amino acid sequence ofSEQ ID NO: 93. In some embodiments, the molecular payload is aDMPK-targeting oligonucleotide (e.g., a DMPK-targeting oligonucleotidelisted in Table 8 or Table 17).

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 102 or SEQ ID NO: 103 and a light chain comprising the aminoacid sequence of SEQ ID NO: 95. In some embodiments, the molecularpayload is a DMPK-targeting oligonucleotide (e.g., a DMPK-targetingoligonucleotide listed in Table 8 or Table 17).

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked via a lysine to the 5′ end of anoligonucleotide (e.g., a DMPK-targeting oligonucleotide), wherein theanti-TfR antibody comprises a heavy chain comprising the amino acidsequence of SEQ ID NO: 84 and a light chain comprising the amino acidsequence of in SEQ ID NO: 85; wherein the complex has the structure of:

wherein n is 3 and m is 4, optionally wherein the DMPK-targetingoligonucleotide (e.g., a gapmer) comprises at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 consecutive nucleotides (e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) of any one of theoligonucleotides listed in Table 8 or Table 17 (e.g., any one of SEQ IDNOs: 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212,215, 218, 222, 248, and 264).

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked via a lysine to the 5′ end of anoligonucleotide (e.g., a DMPK-targeting oligonucleotide, wherein theanti-TfR antibody comprises a heavy chain comprising the amino acidsequence of SEQ ID NO: 86 and a light chain comprising the amino acidsequence of in SEQ ID NO: 85; wherein the complex has the structure of:

wherein n is 3 and m is 4, optionally wherein the DMPK-targetingoligonucleotide (e.g., a gapmer) comprises at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 consecutive nucleotides (e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) of any one of theoligonucleotides listed in Table 8 or Table 17 (e.g., any one of SEQ IDNOs: 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212,215, 218, 222, 248, and 264). In some embodiments, the complex describedherein comprises an anti-TfR antibody covalently linked via a lysine tothe 5′ end of an oligonucleotide (e.g., a DMPK-targetingoligonucleotide), wherein the anti-TfR antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 87 and a light chaincomprising the amino acid sequence of in SEQ ID NO: 85; wherein thecomplex has the structure of:

wherein n is 3 and m is 4, optionally wherein the DMPK-targetingoligonucleotide (e.g., a gapmer) comprises at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 consecutive nucleotides (e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) of any one of theoligonucleotides listed in Table 8 or Table 17 (e.g., any one of SEQ IDNOs: 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212,215, 218, 222, 248, and 264).

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked via a lysine to the 5′ end of anoligonucleotide (e.g., a DMPK-targeting oligonucleotide), wherein theanti-TfR antibody comprises a heavy chain comprising the amino acidsequence of SEQ ID NO: 88 and a light chain comprising the amino acidsequence of in SEQ ID NO: 89; wherein the complex has the structure of:

wherein n is 3 and m is 4, optionally wherein the DMPK-targetingoligonucleotide (e.g., a gapmer) comprises at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 consecutive nucleotides (e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) of any one of theoligonucleotides listed in Table 8 or Table 17 (e.g., any one of SEQ IDNOs: 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212,215, 218, 222, 248, and 264).

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked via a lysine to the 5′ end of anoligonucleotide (e.g., a DMPK-targeting oligonucleotide), wherein theanti-TfR antibody comprises a heavy chain comprising the amino acidsequence of SEQ ID NO: 88 and a light chain comprising the amino acidsequence of in SEQ ID NO: 90; wherein the complex has the structure of:

wherein n is 3 and m is 4, optionally wherein the DMPK-targetingoligonucleotide (e.g., a gapmer) comprises at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 consecutive nucleotides (e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) of any one of theoligonucleotides listed in Table 8 or Table 17 (e.g., any one of SEQ IDNOs: 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212,215, 218, 222, 248, and 264).

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked via a lysine to the 5′ end of anoligonucleotide (e.g., a DMPK-targeting oligonucleotide), wherein theanti-TfR antibody comprises a heavy chain comprising the amino acidsequence of SEQ ID NO: 91 and a light chain comprising the amino acidsequence of in SEQ ID NO: 89; wherein the complex has the structure of:

wherein n is 3 and m is 4, optionally wherein the DMPK-targetingoligonucleotide (e.g., a gapmer) comprises at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 consecutive nucleotides (e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) of any one of theoligonucleotides listed in Table 8 or Table 17 (e.g., any one of SEQ IDNOs: 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212,215, 218, 222, 248, and 264).

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked via a lysine to the 5′ end of anoligonucleotide (e.g., a DMPK-targeting oligonucleotide), wherein theanti-TfR antibody comprises a heavy chain comprising the amino acidsequence of SEQ ID NO: 91 and a light chain comprising the amino acidsequence of in SEQ ID NO: 90; wherein the complex has the structure of:

wherein n is 3 and m is 4, optionally wherein the DMPK-targetingoligonucleotide (e.g., a gapmer) comprises at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 consecutive nucleotides (e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) of any one of theoligonucleotides listed in Table 8 or Table 17 (e.g., any one of SEQ IDNOs: 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212,215, 218, 222, 248, and 264).

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked via a lysine to the 5′ end of anoligonucleotide (e.g., a DMPK-targeting oligonucleotide), wherein theanti-TfR antibody comprises a heavy chain comprising the amino acidsequence of SEQ ID NO: 92 and a light chain comprising the amino acidsequence of in SEQ ID NO: 93; wherein the complex has the structure of:

wherein n is 3 and m is 4, optionally wherein the DMPK-targetingoligonucleotide (e.g., a gapmer) comprises at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 consecutive nucleotides (e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) of any one of theoligonucleotides listed in Table 8 or Table 17 (e.g., any one of SEQ IDNOs: 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212,215, 218, 222, 248, and 264).

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked via a lysine to the 5′ end of anoligonucleotide (e.g., a DMPK-targeting oligonucleotide), wherein theanti-TfR antibody comprises a heavy chain comprising the amino acidsequence of SEQ ID NO: 94 and a light chain comprising the amino acidsequence of in SEQ ID NO: 95; wherein the complex has the structure of:

wherein n is 3 and m is 4, optionally wherein the DMPK-targetingoligonucleotide (e.g., a gapmer) comprises at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 consecutive nucleotides (e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) of any one of theoligonucleotides listed in Table 8 or Table 17 (e.g., any one of SEQ IDNOs: 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212,215, 218, 222, 248, and 264).

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked via a lysine to the 5′ end of anoligonucleotide (e.g., a DMPK-targeting oligonucleotide), wherein theanti-TfR antibody comprises a heavy chain comprising the amino acidsequence of SEQ ID NO: 92 and a light chain comprising the amino acidsequence of in SEQ ID NO: 95; wherein the complex has the structure of:

wherein n is 3 and m is 4, optionally wherein the DMPK-targetingoligonucleotide (e.g., a gapmer) comprises at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 consecutive nucleotides (e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) of any one of theoligonucleotides listed in Table 8 or Table 17 (e.g., any one of SEQ IDNOs: 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212,215, 218, 222, 248, and 264).

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide(e.g., a DMPK-targeting oligonucleotide), wherein the anti-TfR Fabcomprises a VH comprising the amino acid sequence of SEQ ID NO: 69 and aVL comprising the amino acid sequence of in SEQ ID NO: 70; wherein thecomplex has the structure of:

wherein n is 3 and m is 4, optionally wherein the DMPK-targetingoligonucleotide (e.g., a gapmer) comprises at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 consecutive nucleotides (e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) of any one of theoligonucleotides listed in Table 8 or Table 17 (e.g., any one of SEQ IDNOs: 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212,215, 218, 222, 248, and 264).

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide(e.g., a DMPK-targeting oligonucleotide), wherein the anti-TfR Fabcomprises a VH comprising the amino acid sequence of SEQ ID NO: 71 and aVL comprising the amino acid sequence of in SEQ ID NO: 70; wherein thecomplex has the structure of:

wherein n is 3 and m is 4, optionally wherein the DMPK-targetingoligonucleotide (e.g., a gapmer) comprises at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 consecutive nucleotides (e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) of any one of theoligonucleotides listed in Table 8 or Table 17 (e.g., any one of SEQ IDNOs: 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212,215, 218, 222, 248, and 264).

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide(e.g., a DMPK-targeting oligonucleotide), wherein the anti-TfR Fabcomprises a VH comprising the amino acid sequence of SEQ ID NO: 72 and aVL comprising the amino acid sequence of in SEQ ID NO: 70; wherein thecomplex has the structure of:

wherein n is 3 and m is 4, optionally wherein the DMPK-targetingoligonucleotide (e.g., a gapmer) comprises at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 consecutive nucleotides (e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) of any one of theoligonucleotides listed in Table 8 or Table 17 (e.g., any one of SEQ IDNOs: 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212,215, 218, 222, 248, and 264).

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide(e.g., a DMPK-targeting oligonucleotide), wherein the anti-TfR Fabcomprises a VH comprising the amino acid sequence of SEQ ID NO: 73 and aVL comprising the amino acid sequence of in SEQ ID NO: 74; wherein thecomplex has the structure of:

wherein n is 3 and m is 4, optionally wherein the DMPK-targetingoligonucleotide (e.g., a gapmer) comprises at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 consecutive nucleotides (e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) of any one of theoligonucleotides listed in Table 8 or Table 17 (e.g., any one of SEQ IDNOs: 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212,215, 218, 222, 248, and 264).

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide(e.g., a DMPK-targeting oligonucleotide), wherein the anti-TfR Fabcomprises a VH comprising the amino acid sequence of SEQ ID NO: 73 and aVL comprising the amino acid sequence of in SEQ ID NO: 75; wherein thecomplex has the structure of:

wherein n is 3 and m is 4, optionally wherein the DMPK-targetingoligonucleotide (e.g., a gapmer) comprises at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 consecutive nucleotides (e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) of any one of theoligonucleotides listed in Table 8 or Table 17 (e.g., any one of SEQ IDNOs: 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212,215, 218, 222, 248, and 264).

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide(e.g., a DMPK-targeting oligonucleotide), wherein the anti-TfR Fabcomprises a VH comprising the amino acid sequence of SEQ ID NO: 76 and aVL comprising the amino acid sequence of in SEQ ID NO: 74; wherein thecomplex has the structure of:

wherein n is 3 and m is 4, optionally wherein the DMPK-targetingoligonucleotide (e.g., a gapmer) comprises at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 consecutive nucleotides (e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) of any one of theoligonucleotides listed in Table 8 or Table 17 (e.g., any one of SEQ IDNOs: 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212,215, 218, 222, 248, and 264).

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide(e.g., a DMPK-targeting oligonucleotide), wherein the anti-TfR Fabcomprises a VH comprising the amino acid sequence of SEQ ID NO: 76 and aVL comprising the amino acid sequence of in SEQ ID NO: 75; wherein thecomplex has the structure of:

wherein n is 3 and m is 4, optionally wherein the DMPK-targetingoligonucleotide (e.g., a gapmer) comprises at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 consecutive nucleotides (e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) of any one of theoligonucleotides listed in Table 8 or Table 17 (e.g., any one of SEQ IDNOs: 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212,215, 218, 222, 248, and 264).

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide(e.g., a DMPK-targeting oligonucleotide), wherein the anti-TfR Fabcomprises a VH comprising the amino acid sequence of SEQ ID NO: 77 and aVL comprising the amino acid sequence of in SEQ ID NO: 78; wherein thecomplex has the structure of:

wherein n is 3 and m is 4, optionally wherein the DMPK-targetingoligonucleotide (e.g., a gapmer) comprises at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 consecutive nucleotides (e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) of any one of theoligonucleotides listed in Table 8 or Table 17 (e.g., any one of SEQ IDNOs: 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212,215, 218, 222, 248, and 264).

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide(e.g., a DMPK-targeting oligonucleotide), wherein the anti-TfR Fabcomprises a VH comprising the amino acid sequence of SEQ ID NO: 79 and aVL comprising the amino acid sequence of in SEQ ID NO: 80; wherein thecomplex has the structure of:

wherein n is 3 and m is 4, optionally wherein the DMPK-targetingoligonucleotide (e.g., a gapmer) comprises at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 consecutive nucleotides (e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) of any one of theoligonucleotides listed in Table 8 or Table 17 (e.g., any one of SEQ IDNOs: 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212,215, 218, 222, 248, and 264).

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide(e.g., a DMPK-targeting oligonucleotide), wherein the anti-TfR Fabcomprises a VH comprising the amino acid sequence of SEQ ID NO: 77 and aVL comprising the amino acid sequence of in SEQ ID NO: 80; wherein thecomplex has the structure of:

wherein n is 3 and m is 4, optionally wherein the DMPK-targetingoligonucleotide (e.g., a gapmer) comprises at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 consecutive nucleotides (e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) of any one of theoligonucleotides listed in Table 8 or Table 17 (e.g., any one of SEQ IDNOs: 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212,215, 218, 222, 248, and 264).

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide(e.g., a DMPK-targeting oligonucleotide), wherein the anti-TfR Fabcomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:97 and a light chain comprising the amino acid sequence of in SEQ ID NO:85; wherein the complex has the structure of:

wherein n is 3 and m is 4, optionally wherein the DMPK-targetingoligonucleotide (e.g., a gapmer) comprises at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 consecutive nucleotides (e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) of any one of theoligonucleotides listed in Table 8 or Table 17 (e.g., any one of SEQ IDNOs: 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212,215, 218, 222, 248, and 264).

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide(e.g., a DMPK-targeting oligonucleotide), wherein the anti-TfR Fabcomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:98 and a light chain comprising the amino acid sequence of in SEQ ID NO:85; wherein the complex has the structure of:

wherein n is 3 and m is 4, optionally wherein the DMPK-targetingoligonucleotide (e.g., a gapmer) comprises at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 consecutive nucleotides (e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) of any one of theoligonucleotides listed in Table 8 or Table 17 (e.g., any one of SEQ IDNOs: 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212,215, 218, 222, 248, and 264).

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide(e.g., a DMPK-targeting oligonucleotide), wherein the anti-TfR Fabcomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:99 and a light chain comprising the amino acid sequence of in SEQ ID NO:85; wherein the complex has the structure of:

wherein n is 3 and m is 4, optionally wherein the DMPK-targetingoligonucleotide (e.g., a gapmer) comprises at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 consecutive nucleotides (e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) of any one of theoligonucleotides listed in Table 8 or Table 17 (e.g., any one of SEQ IDNOs: 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212,215, 218, 222, 248, and 264).

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide(e.g., a DMPK-targeting oligonucleotide), wherein the anti-TfR Fabcomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:100 and a light chain comprising the amino acid sequence of in SEQ IDNO: 89; wherein the complex has the structure of:

wherein n is 3 and m is 4, optionally wherein the DMPK-targetingoligonucleotide (e.g., a gapmer) comprises at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 consecutive nucleotides (e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) of any one of theoligonucleotides listed in Table 8 or Table 17 (e.g., any one of SEQ IDNOs: 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212,215, 218, 222, 248, and 264).

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide(e.g., a DMPK-targeting oligonucleotide), wherein the anti-TfR Fabcomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:100 and a light chain comprising the amino acid sequence of in SEQ IDNO: 90; wherein the complex has the structure of:

wherein n is 3 and m is 4, optionally wherein the DMPK-targetingoligonucleotide (e.g., a gapmer) comprises at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 consecutive nucleotides (e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) of any one of theoligonucleotides listed in Table 8 or Table 17 (e.g., any one of SEQ IDNOs: 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212,215, 218, 222, 248, and 264).

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide(e.g., a DMPK-targeting oligonucleotide), wherein the anti-TfR Fabcomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:101 and a light chain comprising the amino acid sequence of in SEQ IDNO: 89; wherein the complex has the structure of:

wherein n is 3 and m is 4, optionally wherein the DMPK-targetingoligonucleotide (e.g., a gapmer) comprises at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 consecutive nucleotides (e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) of any one of theoligonucleotides listed in Table 8 or Table 17 (e.g., any one of SEQ IDNOs: 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212,215, 218, 222, 248, and 264).

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide(e.g., a DMPK-targeting oligonucleotide), wherein the anti-TfR Fabcomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:101 and a light chain comprising the amino acid sequence of in SEQ IDNO: 90; wherein the complex has the structure of:

wherein n is 3 and m is 4, optionally wherein the DMPK-targetingoligonucleotide (e.g., a gapmer) comprises at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 consecutive nucleotides (e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) of any one of theoligonucleotides listed in Table 8 or Table 17 (e.g., any one of SEQ IDNOs: 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212,215, 218, 222, 248, and 264).

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide(e.g., a DMPK-targeting oligonucleotide), wherein the anti-TfR Fabcomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:102 and a light chain comprising the amino acid sequence of in SEQ IDNO: 93; wherein the complex has the structure of:

wherein n is 3 and m is 4, optionally wherein the DMPK-targetingoligonucleotide (e.g., a gapmer) comprises at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 consecutive nucleotides (e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) of any one of theoligonucleotides listed in Table 8 or Table 17 (e.g., any one of SEQ IDNOs: 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212,215, 218, 222, 248, and 264).

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide(e.g., a DMPK-targeting oligonucleotide), wherein the anti-TfR Fabcomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:103 and a light chain comprising the amino acid sequence of in SEQ IDNO: 95; wherein the complex has the structure of:

wherein n is 3 and m is 4, optionally wherein the DMPK-targetingoligonucleotide (e.g., a gapmer) comprises at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 consecutive nucleotides (e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) of any one of theoligonucleotides listed in Table 8 or Table 17 (e.g., any one of SEQ IDNOs: 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212,215, 218, 222, 248, and 264).

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide(e.g., a DMPK-targeting oligonucleotide), wherein the anti-TfR Fabcomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:102 and a light chain comprising the amino acid sequence of in SEQ IDNO: 95; wherein the complex has the structure of:

wherein n is 3 and m is 4, optionally wherein the DMPK-targetingoligonucleotide (e.g., a gapmer) comprises at least 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 consecutive nucleotides (e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) of any one of theoligonucleotides listed in Table 8 or Table 17 (e.g., any one of SEQ IDNOs: 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212,215, 218, 222, 248, and 264).

In some embodiments, in any one of the examples of complexes describedherein, L1 is any one of the spacers described herein.

In some embodiments, L1 is:

wherein the piperazine moiety links to the oligonucleotide, wherein L2is

In some embodiments, L1 is:

wherein the piperazine moiety links to the oligonucleotide.

In some embodiments, L1 is

In some embodiments, L1 is linked to a 5′ phosphate of theoligonucleotide.

In some embodiments, L1 is optional (e.g., need not be present).

III. Formulations

Complexes provided herein may be formulated in any suitable manner.Generally, complexes provided herein are formulated in a manner suitablefor pharmaceutical use. For example, complexes can be delivered to asubject using a formulation that minimizes degradation, facilitatesdelivery and/or (e.g., and) uptake, or provides another beneficialproperty to the complexes in the formulation. In some embodiments,provided herein are compositions comprising complexes andpharmaceutically acceptable carriers. Such compositions can be suitablyformulated such that when administered to a subject, either into theimmediate environment of a target cell or systemically, a sufficientamount of the complexes enter target muscle cells. In some embodiments,complexes are formulated in buffer solutions such as phosphate-bufferedsaline solutions, liposomes, micellar structures, and capsids.

It should be appreciated that, in some embodiments, compositions mayinclude separately one or more components of complexes provided herein(e.g., muscle-targeting agents, linkers, molecular payloads, orprecursor molecules of any one of them).

In some embodiments, complexes are formulated in water or in an aqueoussolution (e.g., water with pH adjustments). In some embodiments,complexes are formulated in basic buffered aqueous solutions (e.g.,PBS). In some embodiments, formulations as disclosed herein comprise anexcipient. In some embodiments, an excipient confers to a compositionimproved stability, improved absorption, improved solubility and/or(e.g., and) therapeutic enhancement of the active ingredient. In someembodiments, an excipient is a buffering agent (e.g., sodium citrate,sodium phosphate, a tris base, or sodium hydroxide) or a vehicle (e.g.,a buffered solution, petrolatum, dimethyl sulfoxide, or mineral oil).

In some embodiments, a complex or component thereof (e.g.,oligonucleotide or antibody) is lyophilized for extending its shelf-lifeand then made into a solution before use (e.g., administration to asubject). Accordingly, an excipient in a composition comprising acomplex, or component thereof, described herein may be a lyoprotectant(e.g., mannitol, lactose, polyethylene glycol, or polyvinyl pyrolidone),or a collapse temperature modifier (e.g., dextran, ficoll, or gelatin).

In some embodiments, a pharmaceutical composition is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, administration. Typically, the route of administration isintravenous or subcutaneous.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. The carrier can be a solvent or dispersionmedium containing, for example, water, ethanol, polyol (for example,glycerol, propylene glycol, and liquid polyethylene glycol, and thelike), and suitable mixtures thereof. In some embodiments, formulationsinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, and sodium chloride in the composition. Sterileinjectable solutions can be prepared by incorporating the complexes in arequired amount in a selected solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization.

In some embodiments, a composition may contain at least about 0.1% ofthe complex, or component thereof, or more, although the percentage ofthe active ingredient(s) may be between about 1% and about 80% or moreof the weight or volume of the total composition. Factors such assolubility, bioavailability, biological half-life, route ofadministration, product shelf life, as well as other pharmacologicalconsiderations will be contemplated by one skilled in the art ofpreparing such pharmaceutical formulations, and as such, a variety ofdosages and treatment regimens may be desirable.

IV. Methods of Use/Treatment

Complexes comprising a muscle-targeting agent covalently linked to amolecular payload as described herein are effective in treating myotonicdystrophy. In some embodiments, complexes are effective in treatingmyotonic dystrophy type 1 (DM1). In some embodiments, DM1 is associatedwith an expansion of a CTG trinucleotide repeat in the 3′ non-codingregion of DMPK. In some embodiments, the nucleotide expansions lead totoxic RNA repeats capable of forming hairpin structures that bindcritical intracellular proteins, e.g., muscleblind-like proteins, withhigh affinity.

In some embodiments, a subject may be a human subject, a non-humanprimate subject, a rodent subject, or any suitable mammalian subject. Insome embodiments, a subject may have myotonic dystrophy. In someembodiments, a subject has a DMPK allele, which may optionally contain adisease-associated repeat. In some embodiments, a subject may have aDMPK allele with an expanded disease-associated-repeat that comprisesabout 2-10 repeat units, about 2-50 repeat units, about 2-100 repeatunits, about 50-1,000 repeat units, about 50-500 repeat units, about50-250 repeat units, about 50-100 repeat units, about 500-10,000 repeatunits, about 500-5,000 repeat units, about 500-2,500 repeat units, about500-1,000 repeat units, or about 1,000-10,000 repeat units. In someembodiments, a subject is suffering from symptoms of DM1, e.g. muscleatrophy or muscle loss. In some embodiments, a subject is not sufferingfrom symptoms of DM1. In some embodiments, subjects have congenitalmyotonic dystrophy.

An aspect of the disclosure includes a method involving administering toa subject an effective amount of a complex as described herein. In someembodiments, an effective amount of a pharmaceutical composition thatcomprises a complex comprising a muscle-targeting agent covalentlylinked to a molecular payload can be administered to a subject in needof treatment. In some embodiments, a pharmaceutical compositioncomprising a complex as described herein may be administered by asuitable route, which may include intravenous administration, e.g., as abolus or by continuous infusion over a period of time. In someembodiments, intravenous administration may be performed byintramuscular, intraperitoneal, intracerebrospinal, subcutaneous,intra-articular, intrasynovial, or intrathecal routes. In someembodiments, a pharmaceutical composition may be in solid form, aqueousform, or a liquid form. In some embodiments, an aqueous or liquid formmay be nebulized or lyophilized. In some embodiments, a nebulized orlyophilized form may be reconstituted with an aqueous or liquidsolution.

Compositions for intravenous administration may contain various carrierssuch as vegetable oils, dimethylactamide, dimethyformamide, ethyllactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols(glycerol, propylene glycol, liquid polyethylene glycol, and the like).For intravenous injection, water soluble antibodies can be administeredby the drip method, whereby a pharmaceutical formulation containing theantibody and a physiologically acceptable excipients is infused.Physiologically acceptable excipients may include, for example, 5%dextrose, 0.9% saline, Ringer's solution or other suitable excipients.Intramuscular preparations, e.g., a sterile formulation of a suitablesoluble salt form of the antibody, can be dissolved and administered ina pharmaceutical excipient such as Water-for-Injection, 0.9% saline, or5% glucose solution.

In some embodiments, a pharmaceutical composition that comprises acomplex comprising a muscle-targeting agent covalently linked to amolecular payload is administered via site-specific or local deliverytechniques. Examples of these techniques include implantable depotsources of the complex, local delivery catheters, site specificcarriers, direct injection, or direct application.

In some embodiments, a pharmaceutical composition that comprises acomplex comprising a muscle-targeting agent covalently linked to amolecular payload is administered at an effective concentration thatconfers therapeutic effect on a subject. Effective amounts vary, asrecognized by those skilled in the art, depending on the severity of thedisease, unique characteristics of the subject being treated, e.g. age,physical conditions, health, or weight, the duration of the treatment,the nature of any concurrent therapies, the route of administration andrelated factors. These related factors are known to those in the art andmay be addressed with no more than routine experimentation. In someembodiments, an effective concentration is the maximum dose that isconsidered to be safe for the patient. In some embodiments, an effectiveconcentration will be the lowest possible concentration that providesmaximum efficacy.

Empirical considerations, e.g. the half-life of the complex in asubject, generally will contribute to determination of the concentrationof pharmaceutical composition that is used for treatment. The frequencyof administration may be empirically determined and adjusted to maximizethe efficacy of the treatment.

Generally, for administration of any of the complexes described herein,an initial candidate dosage may be about 1 to 100 mg/kg, or more,depending on the factors described above, e.g. safety or efficacy. Insome embodiments, a treatment will be administered once. In someembodiments, a treatment will be administered daily, biweekly, weekly,bimonthly, monthly, or at any time interval that provide maximumefficacy while minimizing safety risks to the subject. Generally, theefficacy and the treatment and safety risks may be monitored throughoutthe course of treatment.

In some embodiments, an initial candidate dosage is about 1-50, 1-25,1-10, 1-5, 5-100, 5-50, 5-25, 5-10, 10-100, 10-75, 10-50, 10-25, 10-20,25-100, 25-75, or 25-50 mg/kg. In some embodiments, an initial candidatedosage is about 1-20, 1-15, 1-10, 1-5, 1-3, 1-2, 5-20, 5-15, or 5-10mg/kg. In some embodiments, an initial candidate dosage is about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 mg/kg.

The efficacy of treatment may be assessed using any suitable methods. Insome embodiments, the efficacy of treatment may be assessed byevaluation of observation of symptoms associated with DM1, e.g. muscleatrophy or muscle weakness, through measures of a subject'sself-reported outcomes, e.g. mobility, self-care, usual activities,pain/discomfort, and anxiety/depression, or by quality-of-lifeindicators, e.g. lifespan.

In some embodiments, a pharmaceutical composition that comprises acomplex comprising a muscle-targeting agent covalently linked to amolecular payload described herein is administered to a subject at aneffective concentration sufficient to inhibit activity or expression ofa target gene by at least 10%, at least 20%, at least 30%, at least 40%,at least 50%, at least 60%, at least 70%, at least 80%, at least 90% orat least 95% relative to a control, e.g. baseline level of geneexpression prior to treatment.

In some embodiments, a single dose or administration of a pharmaceuticalcomposition that comprises a complex comprising a muscle-targeting agentcovalently linked to a molecular payload described herein to a subjectis sufficient to inhibit activity or expression of a target gene for atleast 1-5, 1-10, 5-15, 10-20, 15-30, 20-40, 25-50, or more days. In someembodiments, a single dose or administration of a pharmaceuticalcomposition that comprises a complex comprising a muscle-targeting agentcovalently linked to a molecular payload described herein to a subjectis sufficient to inhibit activity or expression of a target gene for atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, or 24 weeks. Insome embodiments, a single dose or administration of a pharmaceuticalcomposition that comprises a complex comprising a muscle-targeting agentcovalently linked to a molecular payload described herein to a subjectis sufficient to inhibit activity or expression of a target gene for atleast 1-5, 1-10, 2-5, 2-10, 4-8, 4-12, 5-10, 5-12, 5-15, 8-12, 8-15,10-12, 10-15, 10-20, 12-15, 12-20, 15-20, or 15-25 weeks. In someembodiments, a single dose or administration of a pharmaceuticalcomposition that comprises a complex comprising a muscle-targeting agentcovalently linked to a molecular payload described herein to a subjectis sufficient to inhibit activity or expression of a target gene for atleast 1, 2, 3, 4, 5, or 6 months.

In some embodiments, a single dose or administration of a pharmaceuticalcomposition that comprises a complex comprising a muscle-targeting agentcovalently linked to a molecular payload described herein to a subjectpersists or remains in the subject for at least 1-5, 1-10, 5-15, 10-20,15-30, 20-40, 25-50, or more days. In some embodiments, a single dose oradministration of a pharmaceutical composition that comprises a complexcomprising a muscle-targeting agent covalently linked to a molecularpayload described herein to a subject persists or remains in the subjectfor at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, or 24 weeks.In some embodiments, a single dose or administration of a pharmaceuticalcomposition that comprises a complex comprising a muscle-targeting agentcovalently linked to a molecular payload described herein to a subjectpersists or remains in the subject for at least 1-5, 1-10, 2-5, 2-10,4-8, 4-12, 5-10, 5-12, 5-15, 8-12, 8-15, 10-12, 10-15, 10-20, 12-15,12-20, 15-20, or 15-25 weeks. In some embodiments, a single dose oradministration of a pharmaceutical composition that comprises a complexcomprising a muscle-targeting agent covalently linked to a molecularpayload described herein to a subject persists or remains in the subjectfor at least 1, 2, 3, 4, 5, or 6 months.

In some embodiments, multiple doses or administrations of apharmaceutical composition that comprises a complex comprising amuscle-targeting agent covalently linked to a molecular payloaddescribed herein are delivered to a subject. In some embodiments,multiple doses of a pharmaceutical composition comprise delivering 2, 3,4, 5, 6, 7, 8, 9, or 10 doses to a subject. In some embodiments,multiple doses of a pharmaceutical composition comprise delivering adose to a subject every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, or 16 weeks. In some embodiments, multiple doses of a pharmaceuticalcomposition comprise delivering a dose to a subject once every 4 weeks.In some embodiments, multiple doses of a pharmaceutical compositioncomprise delivering a dose to a subject once every 1-10, 2-5, 2-10, 4-8,4-12, 5-10, 5-12, 5-15, 8-12, 8-16, 10-12, 10-15, 10-20, 12-15, 12-20,15-20, or 15-25 weeks. In some embodiments, multiple doses of apharmaceutical composition comprise delivering a dose to a subject on abiweekly (i.e., every two weeks), bimonthly (i.e., every two months), orquarterly schedule (i.e., every twelve weeks).

In some embodiments, a single dose or administration is about 1-50,1-25, 1-10, 1-15, 1-5, 5-100, 5-50, 5-25, 5-10, 10-100, 10-75, 10-50,10-25, 10-20, 25-100, 25-75, or 25-50 mg/kg. In some embodiments, asingle dose or administration is about 1-20, 1-15, 1-10, 1-5, 1-3, 1-2,5-20, 5-15, or 5-10 mg/kg. In some embodiments, a single dose oradministration is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, or 20 mg/kg.

In some embodiments, a pharmaceutical composition that comprises acomplex comprising a muscle-targeting agent covalently linked to amolecular payload described herein is delivered to a subject every 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks. In someembodiments, a pharmaceutical composition that comprises a complexcomprising a muscle-targeting agent covalently linked to a molecularpayload described herein is delivered to a subject every 1-10, 2-5,2-10, 4-8, 4-12, 5-10, 5-12, 5-15, 8-12, 8-16, 9-15, 10-12, 10-14,10-15, 10-20, 11-13, 11-15, 12-15, 12-16, 12-20, 15-20, or 15-25 weeks.In some embodiments, a pharmaceutical composition that comprises acomplex comprising a muscle-targeting agent covalently linked to amolecular payload described herein is delivered to a subject on abiweekly (i.e., every two weeks), bimonthly (i.e., every two months), orquarterly schedule (i.e., every twelve weeks).

In some embodiments, a pharmaceutical composition that comprises acomplex comprising a muscle-targeting agent covalently linked to amolecular payload described herein at a concentration of 1-15 mg/kg ofRNA is delivered to a subject every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, or 16 weeks. In some embodiments, a pharmaceuticalcomposition that comprises a complex comprising a muscle-targeting agentcovalently linked to a molecular payload described herein at aconcentration of 1-15 mg/kg of RNA is delivered to a subject every 1-10,2-5, 2-10, 4-8, 4-12, 5-10, 5-12, 5-15, 8-12, 8-16, 9-15, 10-12, 10-14,10-15, 10-20, 11-13, 11-15, 12-15, 12-16, 12-20, 15-20, or 15-25 weeks.In some embodiments, a pharmaceutical composition that comprises acomplex comprising a muscle-targeting agent covalently linked to amolecular payload described herein at a concentration of 1-15 mg/kg ofRNA is delivered to a subject on a biweekly (i.e., every two weeks),bimonthly (i.e., every two months), or quarterly schedule (i.e., everytwelve weeks).

In some embodiments, a pharmaceutical composition may comprise more thanone complex comprising a muscle-targeting agent covalently linked to amolecular payload. In some embodiments, a pharmaceutical composition mayfurther comprise any other suitable therapeutic agent for treatment of asubject, e.g. a human subject having DM1. In some embodiments, the othertherapeutic agents may enhance or supplement the effectiveness of thecomplexes described herein. In some embodiments, the other therapeuticagents may function to treat a different symptom or disease than thecomplexes described herein.

EXAMPLES Example 1: Targeting DMPK with Transfected AntisenseOligonucleotides

A gapmer antisense oligonucleotide that targets both wild-type andmutant alleles of DMPK (ASO300) was tested in vitro for its ability toreduce expression levels of DMPK in an immortalized cell line. Briefly,Hepa 1-6 cells were transfected with ASO300 (100 nM) formulated withLipofectamine 2000. DMPK expression levels were evaluated 72 hoursfollowing transfection. A control experiment was also performed in whichvehicle (phosphate-buffered saline) was delivered to Hepa 1-6 cells inculture and the cells were maintained for 72 hours. As shown in FIG. 1 ,it was found that ASO300 reduced DMPK expression levels by ˜90% comparedwith controls.

Example 2: Targeting DMPK with a Muscle-Targeting Complex

A muscle-targeting complex was generated comprising the DMPK ASO used inExample 1 (ASO300) covalently linked, via a cathepsin cleavable linker,to DTX-A-002 (RI7 217 Fab), an anti-transferrin receptor antibody.

Briefly, a maleimidocaproyl-L-valine-L-citrulline-p-aminobenzyl alcoholp-nitrophenyl carbonate (MC-Val-Cit-PABC-PNP) linker molecule wascoupled to NH₂-C₆-ASO300 using an amide coupling reaction. Excess linkerand organic solvents were removed by gel permeation chromatography. Thepurified Val-Cit-linker-ASO300 was then coupled to a thiol on theanti-transferrin receptor antibody (DTX-A-002).

The product of the antibody coupling reaction was then subjected tohydrophobic interaction chromatography (HIC-HPLC). FIG. 2A shows aresulting HIC-HPLC chromatogram, in which fractions B7-C2 of thechromatogram (denoted by vertical lines) containedantibody-oligonucleotide complexes (referred to as DTX-C-008) comprisingone or two DMPK ASO molecules covalently attached to DTX-A-002, asdetermined by SDS-PAGE. These HIC-HPLC fractions were combined anddensitometry following additional purification confirmed that thissample of DTX-C-008 complexes had an average ASO to antibody ratio (DAR)of 1.48. SDS-PAGE analysis demonstrated that 86.4% of this sample ofDTX-C-008 complexes comprised DTX-A-002 linked to either one or two DMPKASO molecules (FIG. 2B).

Using the same methods as described above, a control complex wasgenerated comprising the DMPK ASO used in Example 1 (ASO300) covalentlylinked via a Val-Cit linker to an IgG2a (Fab) antibody (DTX-C-007).

The purified RI7 217 Fab antibody-ASO complex (DTX-C-008) was thentested for cellular internalization and inhibition of DMPK. Hepa 1-6cells, which have relatively high expression levels of transferrinreceptor, were incubated in the presence of vehicle control, DTX-C-008(100 nM), or DTX-C-007 (100 nM) for 72 hours. After the 72 hourincubation, the cells were isolated and assayed for expression levels ofDMPK (FIG. 3 ). Cells treated with the DTX-C-008 demonstrated areduction in DMPK expression by ˜65% relative to the cells treated withthe vehicle control. Meanwhile, cells treated with the DTX-C-007 hadDMPK expression levels comparable to the vehicle control (no reductionin DMPK expression). These data indicate that the anti-transferrinreceptor antibody of the DTX-C-008 enabled cellular internalization ofthe complex, thereby allowing the DMPK ASO to inhibit expression ofDMPK.

Example 3: Targeting DMPK in Mouse Muscle Tissues with aMuscle-Targeting Complex

The muscle-targeting complex described in Example 2, RI7 217 Fabantibody-ASO complex DTX-C-008, was tested for inhibition of DMPK inmouse tissues. C57BL/6 wild-type mice were intravenously injected with asingle dose of a vehicle control, naked ASO300 (3 mg/kg of ASO),DTX-C-008 (3 mg/kg of ASO, corresponding to 20 mg/kg antibodyconjugate), or DTX-C-007 IgG2a Fab antibody-ASO complex (3 mg/kg of ASO,corresponding to 20 mg/kg antibody conjugate). Naked ASO300, the DMPKASO as described in Example 1, was used as a control. Each experimentalcondition was replicated in three individual C57BL/6 wild-type mice.Following a seven-day period after injection, the mice were euthanizedand segmented into isolated tissue types. Individual tissue samples weresubsequently assayed for expression levels of DMPK (FIGS. 4A-4E and5A-5B).

Mice treated with the DTX-C-008 complex demonstrated a reduction in DMPKexpression in a variety of skeletal, cardiac, and smooth muscle tissues.For example, as shown in FIGS. 4A-4E, DMPK expression levels weresignificantly reduced in gastrocnemius (50% reduction), heart (30%reduction), esophagus (45% reduction), tibialis anterior (47%reduction), and soleus (31% reduction) tissues, relative to the micetreated with the vehicle control. Meanwhile, mice treated with theDTX-C-007 complex had DMPK expression levels comparable to the vehiclecontrol mice and mice treated with naked ASO300 (no reduction in DMPKexpression) for all assayed muscle tissue types.

Mice treated with the DTX-C-008 complex demonstrated no change in DMPKexpression in non-muscle tissues such as spleen and brain tissues (FIGS.5A and 5B).

These data indicate that the anti-transferrin receptor antibody of theDTX-C-008 enabled cellular internalization of the complex intomuscle-specific tissues in an in vivo mouse model, thereby allowing theDMPK ASO to inhibit expression of DMPK. These data further demonstratethat the DTX-C-008 complex is capable of specifically targeting muscletissues.

Example 4: Targeting DMPK in Mouse Muscle Tissues with aMuscle-Targeting Complex

The muscle-targeting complex described in Example 2, RI7 217 Fabantibody-ASO complex DTX-C-008, was tested for dose-dependent inhibitionof DMPK in mouse tissues. C57BL/6 wild-type mice were intravenouslyinjected with a single dose of a vehicle control (phosphate-bufferedsaline, PBS), naked ASO300 (10 mg/kg of ASO), DTX-C-008 (3 mg/kg or 10mg/kg of ASO, wherein 3 mg/kg corresponds to 20 mg/kg antibodyconjugate), or DTX-C-007 IgG2a Fab antibody-ASO complex (3 mg/kg or 10mg/kg of ASO, wherein 3 mg/kg corresponds to 20 mg/kg antibodyconjugate). Naked ASO300, the DMPK ASO as described in Example 1, wasused as a control. Each experimental condition was replicated in fiveindividual C57BL/6 wild-type mice. Following a seven-day period afterinjection, the mice were euthanized and segmented into isolated tissuetypes. Individual tissue samples were subsequently assayed forexpression levels of DMPK (FIGS. 6A-6F).

Mice treated with the DTX-C-008 complex demonstrated a reduction in DMPKexpression in a variety of skeletal muscle tissues. As shown in FIGS.6A-6F, DMPK expression levels were significantly reduced in tibialisanterior (58% and 75% reduction for 3 mg/kg and 10 mg/kg DTX-C-008,respectively), soleus (55% and 66% reduction for 3 mg/kg and 10 mg/kgDTX-C-008, respectively), extensor digitorum longus (EDL) (52% and 72%reduction for 3 mg/kg and 10 mg/kg DTX-C-008, respectively),gastrocnemius (55% and 77% reduction for 3 mg/kg and 10 mg/kg DTX-C-008,respectively), heart (19% and 35% reduction for 3 mg/kg and 10 mg/kgDTX-C-008, respectively), and diaphragm (53% and 70% reduction for 3mg/kg and 10 mg/kg DTX-C-008, respectively) tissues, relative to themice treated with the vehicle control. Notably, all assayed muscletissue types experienced dose-dependent inhibition of DMPK, with greaterreduction in DMPK levels at 10 mg/kg antibody conjugate relative to 3mg/kg antibody conjugate.

Meanwhile, mice treated with the control DTX-C-007 complex had DMPKexpression levels comparable to the vehicle control (no reduction inDMPK expression) for all assayed muscle tissue types. These dataindicate that the anti-transferrin receptor antibody of the DTX-C-008enabled cellular internalization of the complex into muscle-specifictissues in an in vivo mouse model, thereby allowing the DMPK ASO toinhibit expression of DMPK. These data further demonstrate that theDTX-C-008 complex is capable of specifically targeting muscle tissuesfor dose-dependent inhibition of DMPK.

Example 5: Targeting DMPK in Cynomolgus Monkey Muscle Tissues with aMuscle-Targeting Complex

A muscle-targeting complex comprising ASO300 (DTX-C-012), was generatedand purified using methods described in Example 2. DTX-C-012 is acomplex comprising a human anti-transferrin receptor antibody covalentlylinked, via a cathepsin cleavable Val-Cit linker, to ASO300. Theanti-TfR antibody used in DTX-C-012 is cross-reactive with cynomolgusand human TfR1. Following HIC-HPLC purification and additionalpurification, densitometry confirmed that DTX-C-012 had an average ASOto antibody ratio of 1.32, and SDS-PAGE revealed a purity of 92.3%.

DTX-C-012 was tested for inhibition of DMPK in male cynomolgus monkeytissues. Male cynomolgus monkeys (19-31 months; 2-3 kg) wereintravenously injected with a single dose of a saline control, nakedASO300 (10 mg/kg of ASO), or DTX-C-012 (10 mg/kg of ASO) on Day 0. Eachexperimental condition was replicated in three individual malecynomolgus monkeys. On Day 7 after injection, tissue biopsies (includingmuscle tissues) were collected. DMPK mRNA expression levels, ASOdetection assays, serum clinical chemistries, tissue histology, clinicalobservations, and body weights were analyzed. The monkeys wereeuthanized on Day 14.

Significant knockdown (KD) of DMPK mRNA expression using DTX-C-012 wasobserved in soleus, deep flexor, and masseter muscles relative to salinecontrol, with 39% KD, 62% KD, and 41% KD, respectively (FIGS. 7A-7C).Robust knockdown of DMPK mRNA expression by DTX-C-012 was furtherobserved in gastrocnemius (62% KD; FIG. 7D), EDL (29% KD; FIG. 7E),tibialis anterior muscle (23% KD; FIG. 7F), diaphragm (54% KD; FIG. 7G),tongue (43% KD; FIG. 7H), heart muscle (36% KD; FIG. 7L), quadriceps(58% KD; FIG. 7J), bicep (51% KD; FIG. 7K), and deltoid muscles (47% KD;FIG. 7L). Knockdown of DMPK mRNA expression by DTX-C-012 in smoothmuscle was also observed in the intestine, with 63% KD atjejunum-duodenum ends (FIG. 8A) and 70% KD in ileum (FIG. 8B). Notably,naked DMPK ASO300 (i.e., not linked to a muscle-targeting agent) hadminimal effects on DMPK expression levels relative to the vehiclecontrol (i.e., little or no reduction in DMPK expression) for mostassayed muscle tissue types. Monkeys treated with the DTX-C-012 complexdemonstrated no change in DMPK expression in most non-muscle tissues,such as kidney, brain, and spleen tissues (FIGS. 9A-9D). Additionaltissues were examined, as depicted in FIG. 10 , which shows normalizedDMPK mRNA tissue expression levels across several tissue types incynomolgus monkeys. (N=3 male cynomolgus monkeys)

Prior to euthanization, all monkeys were tested for reticulocyte levels,platelet levels, hemoglobin expression, alanine aminotransferase (ALT)expression, aspartate aminotransferase (AST) expression, and blood ureanitrogen (BUN) levels on days 2, 7, and 14 after dosing. As shown inFIG. 12 , monkeys dosed with antibody-oligonucleotide complex had normalreticulocyte levels, platelet levels and hemoglobin expressionthroughout the length of the experiment. Monkeys dosed with DTX-C-012also had normal alanine aminotransferase (ALT) expression, aspartateaminotransferase (AST) expression, and blood urea nitrogen (BUN) levelsthroughout the length of the experiment. These data show that a singledose of a complex comprising ASO300 is safe and tolerated in cynomolgusmonkeys.

These data demonstrate that the anti-transferrin receptor antibody ofthe DTX-C-012 complex enabled cellular internalization of the complexinto muscle-specific tissues in an in vivo cynomolgus monkey model,thereby allowing the DMPK ASO (ASO300) to inhibit expression of DMPK.These data further demonstrate that the DTX-C-012 complex is capable ofspecifically targeting muscle tissues for dose-dependent inhibition ofDMPK without substantially impacting non-muscle tissues. This is directcontrast with the limited ability of naked DMPK ASO300 (not linked to amuscle-targeting agent), to inhibit expression of DMPK in muscle tissuesof an in vivo cynomolgus monkey model.

Example 6: Targeting DMPK in Mouse Muscle Tissues with aMuscle-Targeting Complex

The RI7 217 Fab antibody-ASO muscle-targeting complex described inExample 2, DTX-C-008, was tested for time-dependent inhibition of DMPKin mouse tissues. C57BL/6 wild-type mice were intravenously injectedwith a single dose of a vehicle control (saline), naked ASO300 (10 mg/kgof ASO), or DTX-C-008 (10 mg/kg of ASO) and euthanized after aprescribed period of time, as described in Table 13. Followingeuthanization, the mice were segmented into isolated tissue types andtissue samples were subsequently assayed for expression levels of DMPK(FIGS. 11A-11B).

TABLE 13 Experimental conditions Days after injection Number GroupDosage before euthanization of mice 1 Vehicle (saline) 3 days 3 2Vehicle (saline) 7 days 3 3 Vehicle (saline) 14 days 3 4 Vehicle(saline) 28 days 3 5 ASO300 3 days 3 6 ASO300 7 days 3 7 ASO300 14 days3 8 ASO300 28 days 3 9 DTX-C-008 3 days 3 10 DTX-C-008 7 days 3 11DTX-C-008 14 days 3 12 DTX-C-008 28 days 3

Mice treated with the DTX-C-008 complex demonstrated approximately 50%reduction in DMPK expression in gastrocnemius (FIG. 11A) and tibialisanterior (FIG. 11B) muscles for all of Groups 9-12 (3-28 days betweeninjection and euthanization), relative to vehicle. Mice treated with thenaked ASO300 oligonucleotide did not demonstrate significant reductionin DMPK expression.

These data indicate that the DTX-C-008 complex was capable of providingpersistent reduction in DMPK expression for up to 28 days followingdosage of mice with said DTX-C-008 complex.

Example 7: Evaluation of Antisense Oligonucleotides that Target DMPK inImmortalized Myoblasts

Two hundred and thirty-six oligonucleotides for targeting DMPK weregenerated using in silico analysis. Each individual oligonucleotide wasevaluated for their ability to target DMPK in cellulo at two doses—0.5nM (low dose) and 50 nM (high dose).

Briefly, DM1 C15 immortalized myoblasts were cultured in T-75 flasksuntil near confluency (˜80% confluent). Myoblasts were then disruptedwith trypsin and seeded into 96-well microplates at a density of 50,000cells/well. Cells were allowed to recover overnight before the growthmedia was washed out and replaced with a no-serum media to inducedifferentiation into myotubes. Differentiation proceeded for seven daysprior to treatment with DMPK-targeting oligonucleotides.

On day seven following induction of differentiation, DM1 C15 myotubeswere transfected with an individual oligonucleotide using 0.3 μL ofLipofectamine MessengerMax per well. All oligonucleotides were tested atboth 0.5 nM and 50 nM final concentrations in biological triplicates.After treatment with oligonucleotides, cells were incubated for 72 hoursprior to being harvested for total RNA. cDNA was synthesized from thetotal RNA extracts and qPCR was performed to determine expression levelsof DMPK in technical quadruplicate. All qPCR data were analyzed using atraditional ΔΔCT method and were normalized to a plate-based negativecontrol that comprised cells treated with vehicle control (0.3 μL/wellLipofectamine MessengerMax without any oligonucleotide). Results fromthese experiments are shown in Table 8. ‘Normalized DMPK Remaining’ foreach antisense oligonucleotide in Table 8 refers to the expression levelof DMPK in cell treated with said antisense oligonucleotide relative tothe negative control that comprised cells treated with vehicle control(wherein the expression level of the negative control has beennormalized to equal 1.00)

The majority of tested DMPK-targeting antisense oligonucleotidesdemonstrated a reduction in DMPK expression in differentiated myotubesat both the low and high dose concentrations (0.5 nM and 50 nM,respectively). These data demonstrate that the antisenseoligonucleotides shown in Table 8 are capable of targeting DMPK incellulo, suggesting that muscle-targeting complexes comprising theseantisense oligonucleotides would be capable of targeting DMPK in muscletissues in vivo.

TABLE 8Ability ofDMPK-targeting antisense oligonucleotides to reduce expression of DMPKin cellulo 0.5 nM 50 nM Antisense SEQ DMPK SEQ Normalized PercentNormalized Percent Sequence NO: Target NO: DMPK DMPK DMPK DMPKOligonucleotide ID Sequence ID Remaining Reduction Remaining ReductionGGACGGCCCG 148 GGCAGCAAGC 384 0.42 58.25 0.31 69.30 GCUUGCUGCCCGGGCCGTCC GGGCCCGGAU 149 CAGTCCTGTG 385 0.42 57.97 0.38 61.96CACAGGACUG ATCCGGGCCC CAAACUUGCU 150 GACACTGCTG 386 0.69 31.45 0.4653.93 CAGCAGUGUC AGCAAGTTTG AAACUUGCUC 151 TGACACTGCT 387 0.69 30.850.49 50.69 AGCAGUGUCA GAGCAAGTTT CGGAUGGCCU 152 CGGGAGATGG 388 0.7128.92 0.44 55.57 CCAUCUCCCG AGGCCATCCG CUCGGCCGGA 153 GGGAGCGGAT 3890.71 28.64 0.35 64.75 AUCCGCUCCC TCCGGCCGAG UCUCGGCCGG 154 GGAGCGGATT390 0.72 27.88 0.33 67.46 AAUCCGCUCC CCGGCCGAGA UGCUCAGCAG 155CCTGCTGACA 391 0.73 27.08 0.34 65.78 UGUCAGCAGG CTGCTGAGCA UUGUCGGGUU156 AGGGACATCA 392 0.66 34.16 0.44 55.56 UGAUGUCCCU AACCCGACAAGUUGCGGGUU 157 GGGACATCAA 393 0.67 33.31 0.39 61.07 UGAUGUCCC ACCCGACAACUCCGCCAGGU 158 GCGCGCTTCT 394 0.72 27.99 0.20 80.06 AGAAGCGCGCACCTGGCGGA CAUGGCAUAC 159 CGGGCCAGGT 395 0.68 31.63 0.26 74.03ACCUGGCCCG GTATGCCATG AACUUGCUCA 160 CTGACACTGC 396 0.80 19.81 0.4752.64 GCAGUGUCAG TGAGCAAGTT CAGCUGCGUG 161 GGCGGTGGAT 397 0.81 19.030.32 68.34 AUCCACCGCC CACGCAGCTG CGAAUGUCCG 162 GAGACACTGT 398 0.6040.21 0.36 64.42 ACAGUGUCUC CGGACATTCG GAAGUCGGCC 163 ACATCCGCCT 3990.82 18.36 0.56 44.04 AGGCGGAUGU GGCCGACTTC UGUCGGGUUU 164 CAGGGACATC400 0.70 30.09 0.32 68.14 GAUGUCCCUG AAACCCGACA GGAUGGCCUC 165CCGGGAGATG 401 0.75 24.93 0.39 60.77 CAUCUCCCGG GAGGCCATCC AGGAUGUUGU166 ATCAAACCCG 402 0.76 24.19 0.61 39.48 CGGGUUUGAU ACAACATCCTGUCGGGUUUG 167 ACAGGGACAT 403 0.71 28.89 0.36 64.15 AUGUCCCUGUCAAACCCGAC AAUACUCCAU 168 GTACCTGGTC 404 0.71 28.86 0.48 52.07GACCAGGUAC ATGGAGTATT CUUGUUCAUG 169 CCATGAAGAT 405 0.84 16.06 0.5149.47 AUCUUCAUGG CATGAACAAG UCAGUGCAUC 170 CCACGTTTTGG 406 0.84 15.760.58 42.06 CAAAACGUGG ATGCACTGA CUGUCCCGGA 171 TGGGATGGTC 407 0.64 35.850.49 50.78 GACCAUCCCA TCCGGGACAG GGGCCUGGGA 172 GACAGTGAGG 408 0.6337.19 0.23 76.81 CCUCACUGUC TCCCAGGCCC CCCACGUAAU 173 GTCATGGAGT 4090.72 28.21 0.54 45.94 ACUCCAUGAC ATTACGTGGG CUCUGCCGCA 174 CGGCTGTCCCT410 0.63 37.09 0.06 93.59 GGGACAGCCG GCGGCAGAG CUGUGCACGU 175 CGGCTTGGCT411 0.74 25.67 0.30 70.10 AGCCAAGCCG ACGTGCACAG UGCCCAUCCA 176GGCCCTGACG 412 0.86 13.63 0.67 33.09 CGUCAGGGCC TGGATGGGCA AGCGCCUCCG177 CCTGGCCTATC 413 0.79 21.19 0.38 61.91 AUAGGCCAGG GGAGGCGCTUGUGCACGUA 178 CCGGCTTGGC 414 0.75 24.74 0.25 75.09 GCCAAGCCGGTACGTGCACA GACCAGGUAC 179 AGAACTACCT 415 0.57 42.85 0.29 70.95AGGUAGUUCU GTACCTGGTC CCAUCUCGGC 180 GCGGATTCCG 416 0.79 20.50 0.4059.76 CGGAAUCCGC GCCGAGATGG CAUCUCGGCC 181 AGCGGATTCC 417 0.80 20.210.41 59.40 GGAAUCCGCU GGCCGAGATG UUGCCAUAGG 182 ACGGCGGAGA 418 0.6436.30 0.40 60.12 UCUCCGCCGU CCTATGGCAA ACAGCGGUCC 183 ACATCCTGCT 4190.80 19.94 0.45 55.14 AGCAGGAUGU GGACCGCTGT AAAGCGCCUC 184 TGGCCTATCG420 0.80 19.89 0.38 62.04 CGAUAGGCCA GAGGCGCTTT GCCAAAGAAG 185CACATCCCTTC 421 0.75 24.87 0.44 56.19 AAGGGAUGUG TTCTTTGGC CACGUAAUAC186 TGGTCATGGA 422 0.76 24.40 0.54 46.50 UCCAUGACCA GTATTACGTGAUCUCGGCCG 187 GAGCGGATTC 423 0.88 11.61 0.34 65.98 GAAUCCGCUCCGGCCGAGAT GCUUCAUCUU 188 AGCGGTAGTG 424 0.69 31.44 0.48 51.78CACUACCGCU AAGATGAAGC GCCAUCUCGG 189 CGGATTCCGG 425 0.81 18.56 0.1486.39 CCGGAAUCCG CCGAGATGGC CAGGGACAGC 190 AGTTCCAGCG 426 0.68 32.090.41 58.84 CGCUGGAACU GCTGTCCCTG AUGACAAUCU 191 TACCTGGCGG 427 0.5842.38 0.40 60.47 CCGCCAGGUA AGATTGTCAT GGCCAUGACA 192 TGGCGGAGAT 4280.58 42.38 0.25 75.00 AUCUCCGCCA TGTCATGGCC AUACUCCAUG 193 TGTACCTGGTC429 0.77 23.07 0.43 56.84 ACCAGGUACA ATGGAGTAT GCCUCUGCCU 194 CAACTACGCG430 0.65 35.38 0.19 81.18 CGCGUAGUUG AGGCAGAGGC GAAUGUCCGA 195GGAGACACTG 431 0.70 30.09 0.37 63.41 CAGUGUCUCC TCGGACATTC CGUUCCAUCU196 AGCTGCGGGC 432 0.66 33.74 0.31 68.72 GCCCGCAGCU AGATGGAACGCCUUGUAGUG 197 CAAGATCGTC 433 0.83 17.20 0.34 65.91 GACGAUCUUGCACTACAAGG AUCUCCGCCA 198 CGCTTCTACCT 434 0.58 42.37 0.35 65.50GGUAGAAGCG GGCGGAGAT CUCAGGCUCU 199 CTCACCCGGC 435 0.70 30.13 0.37 63.07GCCGGGUGAG AGAGCCTGAG UGCUUCAUCU 200 GCGGTAGTGA 436 0.71 28.82 0.4060.24 UCACUACCGC AGATGAAGCA GCAGGAUGUU 201 CAAACCCGAC 437 0.56 44.390.22 78.03 GUCGGGUUUG AACATCCTGC GGCCUCAGCC 202 CTGCGGCAGA 438 0.8020.12 0.29 71.28 UCUGCCGCAG GGCTGAGGCC UGUUGUCGGG 203 GGACATCAAA 4390.79 21.00 0.58 42.19 UUUGAUGUCC CCCGACAACA CCACGUAAUA 204 GGTCATGGAG440 0.79 20.84 0.50 50.06 CUCCAUGACC TATTACGTGG CCGUUCCAUC 205GCTGCGGGCA 441 0.68 31.74 0.23 77.46 UGCCCGCAGC GATGGAACGG UUCCCGAGUA206 TCTGCCTGCTT 442 0.69 31.49 0.50 49.81 AGCAGGCAGA ACTCGGGAAUGAUCUUCAU 207 GGTGTATGCC 443 0.72 27.70 0.10 89.68 GGCAUACACCATGAAGATCA AGGGACAGCC 208 CAGTTCCAGC 444 0.71 28.72 0.55 45.34GCUGGAACTG GGCTGTCCCT GGGUUUGAUG 209 TGCACAGGGA 445 0.60 40.12 0.3762.61 UCCCUGUGCA CATCAAACCC UGACAAUCUC 210 CTACCTGGCG 446 0.61 38.860.33 66.56 CGCCAGGUAG GAGATTGTCA CACAGCGGUC 211 CATCCTGCTG 447 0.93 6.620.40 59.58 CAGCAGGAUG GACCGCTGTG GCGUAGAAGG 212 GGGCAGACGC 448 0.6039.53 0.22 77.91 GCGUCUGCCC CCTTCTACGC CUCAGCCUCU 213 TCCCTGCGGC 4490.82 17.86 0.20 79.58 GCCGCAGGGA AGAGGCTGAG GUCUCAGUGC 214 CGTTTTGGATG450 0.81 18.85 0.54 46.13 AUCCAAAACG CACTGAGAC GGACGAUCUU 215 GACCTATGGC451 0.70 29.82 0.51 48.97 GCCAUAGGUC AAGATCGTCC UCAGCAGUGU 216GGACCTGCTG 452 0.67 33.46 0.39 61.11 CAGCAGGUCC ACACTGCTGA GCUCCUGGGC217 GTCTGGCGCC 453 0.91 8.52 0.21 78.79 GGCGCCAGAC GCCCAGGAGC AGCAGGAUGU218 AAACCCGACA 454 0.59 41.05 0.26 74.02 UGUCGGGUUU ACATCCTGCTAUCCGCUCCU 219 CGGCAGTTGC 455 0.87 12.80 0.60 40.06 GCAACUGCCGAGGAGCGGAT AGGAGCAGGG 220 GAGGCGCTTT 456 0.67 33.24 0.38 62.37AAAGCGCCUC CCCTGCTCCT ACACCUGGCC 221 GAAGCAGACG 457 0.67 33.00 0.4555.40 CGUCUGCUUC GGCCAGGTGT CCCAGCGCCC 222 TGTGACTGGT 458 0.62 37.930.32 67.82 ACCAGUCACA GGGCGCTGGG GCUCCCUCUG 223 TTGCTGCAGG 459 0.7426.41 0.30 70.15 CCUGCAGCAA CAGAGGGAGC GCUCAGGCUC 224 TCACCCGGCA 4600.74 25.69 0.39 60.71 UGCCGGGUGA GAGCCTGAGC UUGAUGUCCC 225 TACGTGCACA461 0.74 25.67 0.45 55.13 UGUGCACGUA GGGACATCAA GCCUCAGCCU 226CCTGCGGCAG 462 0.84 16.37 0.54 46.42 CUGCCGCAGG AGGCTGAGGC GGUAGUUCUC227 CTTCCAGGAT 463 0.75 25.48 0.44 56.15 AUCCUGGAAG GAGAACTACCCAGCGCCCAC 228 AGTGTGACTG 464 0.63 37.28 0.35 64.93 CAGUCACACUGTGGGCGCTG CCCAAACUUG 229 CACTGCTGAG 465 0.63 37.02 0.38 61.78CUCAGCAGUG CAAGTTTGGG CUUGCCAUAG 230 CGGCGGAGAC 466 0.73 27.04 0.2971.05 GUCUCCGCCG CTATGGCAAG UACACCUGGC 231 AAGCAGACGG 467 0.69 31.100.43 57.43 CCGUCUGCUU GCCAGGTGTA CCAGCGCCCA 232 GTGTGACTGG 468 0.6436.17 0.29 70.96 CCAGUCACAC TGGGCGCTGG GGCCUCAGCC 233 CTTTCGGCCA 4690.86 14.49 0.35 64.80 UGGCCGAAAG GGCTGAGGCC AAUCUCCGCC 234 GCTTCTACCTG470 0.64 35.85 0.35 65.27 AGGUAGAAGC GCGGAGATT AUGGCAUACA 235 ACGGGCCAGG471 0.86 14.31 0.50 49.63 CCUGGCCCGU TGTATGCCAT CCAUGACAAU 236CCTGGCGGAG 472 0.65 34.53 0.24 76.46 CUCCGCCAGG ATTGTCATGG UCCCCAAACU237 CTGCTGAGCA 473 0.94 5.73 0.55 44.67 UGCUCAGCAG AGTTTGGGGA GAUGUUGUCG238 ACATCAAACC 474 0.90 10.06 0.58 42.42 GGUUUGAUGU CGACAACATCGUUUGCCCAU 239 CCTGACGTGG 475 0.66 34.36 0.46 54.49 CCACGUCAGGATGGGCAAAC CGGACGGCCC 240 GCAGCAAGCC 476 0.95 5.42 0.70 30.41 GGCUUGCUGCGGGCCGTCCG CUCCGCCAGG 241 CGCGCTTCTAC 477 0.70 30.22 0.22 78.14UAGAAGCGCG CTGGCGGAG GUACAGGUAG 242 AGGATGAGAA 478 0.68 31.52 0.34 65.57UUCUCAUCCU CTACCTGTAC AGGGCGUCUG 243 GTTCTATGGG 479 0.87 13.23 0.4158.98 CCCAUAGAAC CAGACGCCCT UGGCCACAGC 244 CTGCTGGACC 480 0.70 29.590.31 69.44 GGUCCAGCAG GCTGTGGCCA CGUAGUUGAC 245 AACTTCGCCA 481 0.7525.26 0.38 61.52 UGGCGAAGUU GTCAACTACG UCUGCCGCAG 246 GCGGCTGTCC 4820.77 22.97 0.18 82.10 GGACAGCCGC CTGCGGCAGA AAGCGCCUCC 247 CTGGCCTATC483 0.91 8.91 0.56 43.93 GAUAGGCCAG GGAGGCGCTT GACAGAACAA 248CTGTTCGCCGT 484 0.79 21.41 0.30 70.49 CGGCGAACAG TGTTCTGTC GCUCAGCAGU249 ACCTGCTGAC 485 0.71 29.18 0.27 73.46 GUCAGCAGGU ACTGCTGAGCAUGAUCUUCA 250 GTGTATGCCA 486 0.87 12.76 0.60 39.97 UGGCAUACACTGAAGATCAT UUUGCCCAUC 251 CCCTGACGTG 487 0.67 32.79 0.41 59.36CACGUCAGGG GATGGGCAAA ACUUGCUCAG 252 GCTGACACTG 488 0.72 27.84 0.3960.71 CAGUGUCAGC CTGAGCAAGT UGAUGUCCCU 253 CTACGTGCAC 489 0.79 20.580.41 59.00 GUGCACGUAG AGGGACATCA AAAUACCGAG 254 CCCGACATTC 490 0.8911.25 0.49 50.91 GAAUGUCGGG CTCGGTATTT GGCGAAUACA 255 GGGCGCTGGG 4910.80 19.77 0.31 68.72 CCCAGCGCCC TGTATTCGCC AGACAAUAAA 256 TTCCTCGGTAT492 0.71 29.37 0.52 48.20 UACCGAGGAA TTATTGTCT CCCGUCUGCU 257 GTGAAGATGA493 0.80 20.31 0.56 43.97 UCAUCUUCAC AGCAGACGGG CUGCCUGCAG 258GATGGAGTTG 494 0.77 23.10 0.53 46.69 CAACUCCAUC CTGCAGGCAG CCUCAGCCUC259 CCCTGCGGCA 495 0.89 10.87 0.45 55.22 UGCCGCAGGG GAGGCTGAGGGUGUCCGGAA 260 AGCAGGCGAC 496 0.77 22.99 0.26 73.65 GUCGCCUGCUTTCCGGACAC UGCACGUGUG 261 CTGCTTGAGC 497 0.89 10.81 0.36 64.18GCUCAAGCAG CACACGTGCA GACAAUAAAU 262 ATTCCTCGGTA 498 0.71 28.97 0.5247.51 ACCGAGGAAU TTTATTGTC GCCAUGACAA 263 CTGGCGGAGA 499 0.69 30.96 0.1981.00 UCUCCGCCAG TTGTCATGGC GCUGUCCCGG 264 GGGATGGTCT 500 0.77 22.570.34 66.27 AGACCAUCCC CCGGGACAGC CAUGACCAGG 265 ACTACCTGTA 501 0.8119.39 0.41 59.09 UACAGGUAGU CCTGGTCATG AGCGCCCACC 266 GAGTGTGACT 5020.70 30.36 0.36 63.67 AGUCACACUC GGTGGGCGCT UCUCAGUGCA 267 ACGTTTTGGAT503 0.89 10.88 0.49 51.34 UCCAAAACGU GCACTGAGA UUUGGGCAGA 268 AGGCCCTCCA504 0.65 35.14 0.30 70.00 UGGAGGGCCU TCTGCCCAAA GAUGUCCCUG 269GCTACGTGCA 505 0.81 18.99 0.38 62.46 UGCACGUAGC CAGGGACATC CAGCAGUGUC270 GGGACCTGCT 506 0.74 25.67 0.48 51.97 AGCAGGUCCC GACACTGCTGCAUGACAAUC 271 ACCTGGCGGA 507 0.71 29.45 0.29 70.52 UCCGCCAGGUGATTGTCATG ACUUGUUCAU 272 CATGAAGATC 508 0.75 25.47 0.47 52.89GAUCUUCAUG ATGAACAAGT GUGGAAUCCG 273 CCCTTCTACGC 509 0.69 30.55 0.5149.34 CGUAGAAGGG GGATTCCAC UGGCCAUGAC 274 GGCGGAGATT 510 0.70 30.46 0.2772.55 AAUCUCCGCC GTCATGGCCA GGGACAGACA 275 CGGTATTTATT 511 0.73 27.190.49 50.50 AUAAAUACCG GTCTGTCCC CCGCUCCCCA 276 TGAGCAAGTT 512 1.00 0.280.43 56.82 AACUUGCUCA TGGGGAGCGG CGGCUCAGGC 277 ACCCGGCAGA 513 0.8217.97 0.31 69.03 UCUGCCGGGU GCCTGAGCCG GGCUCCUGGG 278 TCTGGCGCCG 5141.00 0.05 0.04 96.23 CGGCGCCAGA CCCAGGAGCC UUUCCCGAGU 279 CTGCCTGCTTA515 0.79 20.69 0.55 44.89 AAGCAGGCAG CTCGGGAAA GGAUGUUGUC 280 CATCAAACCC516 0.96 4.26 0.59 40.81 GGGUUUGAUG GACAACATCC CAGGUAGUUC 281 TCCAGGATGA517 0.74 25.92 0.23 76.71 UCAUCCUGGA GAACTACCTG UGCCCAUAGA 282TATGAAATGT 518 0.92 7.67 0.65 34.56 ACAUUUCAUA TCTATGGGCA UAGUUCUCAU 283GCCTTCCAGG 519 0.83 16.83 0.56 43.88 CCUGGAAGGC ATGAGAACTA AUGUCCCUGU284 GGCTACGTGC 520 0.83 16.78 0.51 49.29 GCACGUAGCC ACAGGGACATCGGGCCCGGA 285 AGTCCTGTGA 521 0.83 17.45 0.33 67.11 UCACAGGACUTCCGGGCCCG UGGACGAUCU 286 ACCTATGGCA 522 0.81 19.20 0.57 42.52UGCCAUAGGU AGATCGTCCA GUUGGCCGGC 287 GGTGGCCCAC 523 1.02 -1.82 0.5643.57 GUGGGCCACC GCCGGCCAAC CUCAGUGCAU 288 CACGTTTTGG 524 0.92  7.650.46 54.26 CCAAAACGUG ATGCACTGAG UCGAAGUUGC 289 ACCGACACAT 525 0.7722.96 0.42 58.15 AUGUGUCGGU GCAACTTCGA UGGAACACGG 290 GCCGGGCCGT 5261.02 -1.90 0.39 60.96 ACGGCCCGGC CCGTGTTCCA CCGAGAGCAG 291 CTCACTTGCGC527 0.84 16.13 0.59 40.93 CGCAAGUGAG TGCTCTCGG UCCUGCAACU 292 CACGTCCGGC528 0.84 16.06 0.55 44.61 GCCGGACGUG AGTTGCAGGA UCACCAACAC 293GGAGAGGGAC 529 0.53 47.12 0.16 84.09 GUCCCUCUCC GTGTTGGTGA UGCCUGCAGC294 GGATGGAGTT 530 0.86 13.99 0.50 49.75 AACUCCAUCC GCTGCAGGCAUUGGCCGGCG 295 TGGTGGCCCA 531 1.03 -3.19 0.56 44.37 UGGGCCACCACGCCGGCCAA GAGCCUCUGC 296 ACTACGCGAG 532 0.81 18.77 0.22 77.78CUCGCGUAGU GCAGAGGCTC AAGGGCGUCU 297 TTCTATGGGC 533 0.87 13.15 0.6534.56 GCCCAUAGAA AGACGCCCTT ACAGACAAUA 298 CCTCGGTATTT 534 1.04 -3.950.26 74.02 AAUACCGAGG ATTGTCTGT GGACAGACAA 299 TCGGTATTTAT 535 0.7722.57 0.47 52.51 UAAAUACCGA TGTCTGTCC ACGUGUGCCU 300 CGGGACCTAG 536 0.8416.47 0.22 77.73 CUAGGUCCCG AGGCACACGT GGCACGAGAC 301 CCGTTGTTCTG 5370.84 16.10 0.32 68.01 AGAACAACGG TCTCGTGCC UGACCAGGUA 302 GAACTACCTG 5380.78 22.00 0.36 63.73 CAGGUAGUUC TACCTGGTCA CUCUGCCGGG 303 GAGGTGCTCA539 0.75 25.25 0.26 74.36 UGAGCACCUC CCCGGCAGAG GACAAUCUCC 304TCTACCTGGC 540 0.76 23.70 0.50 49.82 GCCAGGUAGA GGAGATTGTC UCUCCGCCAG305 GCGCTTCTACC 541 0.80 19.59 0.33 66.52 GUAGAAGCGC TGGCGGAGACUCUGCCUCG 306 GTCAACTACG 542 0.83 16.61 0.09 91.21 CGUAGUUGACCGAGGCAGAG CUUUGGGCAG 307 GGCCCTCCAT 543 0.72 28.06 0.33 67.50AUGGAGGGCC CTGCCCAAAG ACAGGUAGUU 308 CCAGGATGAG 544 0.79 20.51 0.1585.36 CUCAUCCUGG AACTACCTGT CCAAACUUGC 309 ACACTGCTGA 545 0.76 23.640.42 57.70 TCAGCAGUGU GCAAGTTTGG UCGGGUUUGA 310 CACAGGGACA 546 0.7822.49 0.43 57.16 UGUCCCUGUG TCAAACCCGA GGCUUGCUGC 311 GCCTGGGAAG 5471.06 -6.32 0.52 48.15 CUUCCCAGGC GCAGCAAGCC UACAGGUAGU 312 CAGGATGAGA548 0.80 19.83 0.27 72.51 UCUCAUCCUG ACTACCTGTA UUGCCCAUCC 313GCCCTGACGT 549 0.78 22.23 0.33 67.15 ACGUCAGGGC GGATGGGCAA AGGUACAGGU314 GATGAGAACT 550 0.81 18.68 0.41 58.92 AGUUCUCAUC ACCTGTACCTGACAGACAAU 315 CTCGGTATTTA 551 0.82 18.26 0.62 38.07 AAAUACCGAGTTGTCTGTC UAGAACAUUU 316 TTCGCCTATGA 552 0.80 20.23 0.56 43.67CAUAGGCGAA AATGTTCTA AGGGCCUUUU 317 CCTCGCGAAT 553 0.86 13.63 0.34 66.43AUUCGCGAGG AAAAGGCCCT GCCUCGCGUA 318 GCCAGTCAAC 554 0.87 12.98 0.0991.10 GUUGACUGGC TACGCGAGGC CCAGCAGGAU 319 ACCCGACAAC 555 0.60 40.290.10 89.59 GUUGUCGGGU ATCCTGCTGG GUAGUUGACU 320 GAACTTCGCC 556 0.93 7.50 0.55 45.33 GGCGAAGUUC AGTCAACTAC UGCGGAUGGC 321 GGAGATGGAG 5570.60 40.15 0.16 84.43 CUCCAUCUCC GCCATCCGCA ACAAUCUCCG 322 TTCTACCTGGC558 0.81 19.09 0.50 49.75 CCAGGUAGAA GGAGATTGT GCGAAUACAC 323 TGGGCGCTGG559 0.93  6.94 0.30 69.72 CCAGCGCCCA GTGTATTCGC GUAGUUCUCA 324CCTTCCAGGA 560 0.93  7.43 0.45 55.09 UCCUGGAAGG TGAGAACTAC GGCUCAGGCU325 CACCCGGCAG 561 0.93  7.38 0.34 65.82 CUGCCGGGUG AGCCTGAGCCCCAUUCACCA 326 AGGGACGTGT 562 0.61 39.26 0.13 86.83 ACACGUCCCUTGGTGAATGG ACCAGGUACA 327 GAGAACTACC 563 0.84 16.09 0.23 76.96GGUAGUUCUC TGTACCTGGT CTGCAGUUUG 328 CGTGGATGGG 564 1.11 -10.69 0.4060.08 CCCAUCCACG CAAACTGCAG UUGUUCAUGA 329 GCCATGAAGA 565 0.86 14.130.55 45.23 UCUUCAUGGC TCATGAACAA UUGAUGUCCC 330 ACGTGCACAG 566 0.93 6.920.57 43.07 UGUGCACGU GGACATCAAA GCGGUCCAGC 331 ACAACATCCT 567 0.61 38.840.16 83.64 AGGAUGUUGU GCTGGACCGC GUCUAUGGCC 332 AGATTGTCAT 568 1.11-11.00 0.27 73.11 AUGACAAUCU GGCCATAGAC GGAGCAGGGA 333 GGAGGCGCTT 5690.79 21.46 0.12 88.35 AAGCGCCUCC TCCCTGCTCC UGCCUCGCGU 334 CCAGTCAACT570 0.89 11.03 0.12 88.02 AGUUGACUGG ACGCGAGGCA GCGGAUGGCC 335GGGAGATGGA 571 0.79 21.25 0.28 71.77 UCCAUCUCCC GGCCATCCGC UUUCAUAGGC336 GGGTGTATTC 572 0.94 5.56 0.47 53.28 GAAUACACCC GCCTATGAAA GCCUGUCAGC337 CCTCCGACTC 573 0.89 10.81 0.24 75.67 GAGUCGGAGG GCTGACAGGCCCACUUCAGC 338 GGATGAAACA 574 0.78 22.40 0.36 64.20 UGUUUCAUCCGCTGAAGTGG CAUCCGCUCC 339 GGCAGTTGCA 575 0.79 21.04 0.23 76.81UGCAACUGCC GGAGCGGATG UCUAGGGUUC 340 CGCGCTCCCT 576 0.78 21.81 0.1783.22 AGGGAGCGCG GAACCCTAGA CACCAACACG 341 AGGAGAGGGA 577 0.62 37.510.18 81.57 UCCCUCUCCU CGTGTTGGTG CAGGAGCAGG 342 AGGCGCTTTC 578 0.8812.48 0.48 51.82 GAAAGCGCCU CCTGCTCCTG CAAUCUCCGC 343 CTTCTACCTGG 5790.84 15.95 0.51 49.25 CAGGUAGAAG CGGAGATTG AUGUUGUCGG 344 GACATCAAAC 5800.83 16.93 0.47 52.83 GUUUGAUGUC CCGACAACAT CCAUCCGCUC 345 GCAGTTGCAG581 0.80 19.53 0.28 71.62 CUGCAACUGC GAGCGGATGG GCGUCACCUC 346GGCTGAGGCC 582 0.80 20.02 0.19 81.27 GGCCUCAGCC GAGGTGACGC GAGGGCCUUU347 CTCGCGAATA 583 0.92 8.23 0.38 62.21 UAUUCGCGAG AAAGGCCCTC AGCGGCAGAG348 GAGCACCTCT 584 0.80 19.75 0.09 90.71 AGAGGUGCUC CTCTGCCGCTCAUCCAAAAC 349 CCCAATCCAC 585 0.81 19.12 0.22 77.98 GUGGAUUGGGGTTTTGGATG UUGGGCAGAU 350 AAGGCCCTCC 586 0.81 19.08 0.22 78.39GGAGGGCCUU ATCTGCCCAA CCUCUGCCUC 351 TCAACTACGC 587 0.93 7.39 0.15 85.33GCGUAGUUGA GAGGCAGAGG ACAGAACAAC 352 CCTGTTCGCCG 588 0.98 2.07 0.4455.96 GGCGAACAGG TTGTTCTGT CAGGAUGUUG 353 TCAAACCCGA 589 0.83 17.17 0.2179.31 UCGGGUUUGA CAACATCCTG CGGCCUCAGC 354 TGCGGCAGAG 590 0.93 6.71 0.4060.06 CUCUGCCGCA GCTGAGGCCG CAGCAGGAUG 355 AACCCGACAA 591 0.66 34.180.15 84.54 UUGUCGGGUU CATCCTGCTG GCAGAGAGAG 356 CAAGGAGCAC 592 0.8317.29 0.14 85.95 GUGCUCCUUG CTCTCTCTGC UCCAGUUCCA 357 CCCACACCCA 5930.84 15.66 0.22 78.48 UGGGUGUGGG TGGAACTGGA CCUCAGCCUG 358 TTCTTTCGGCC594 0.83 16.83 0.36 63.99 GCCGAAAGAA AGGCTGAGG GGGCCUUUUA 359 CCCTCGCGAA595 0.95 5.11 0.49 50.65 UUCGCGAGGG TAAAAGGCCC GUCGGCCAGG 360 GCCACATCCG596 0.85 15.35 0.25 74.59 CGGAUGUGGC CCTGGCCGAC GCUUGCUGCC 361GGCCTGGGAA 597 0.99 1.14 0.19 81.01 UUCCCAGGCC GGCAGCAAGC GGUCCAGCAG 362CGACAACATC 598 0.68 31.78 0.20 79.93 GAUGUUGUCG CTGCTGGACC CGGAGACCAU363 CTCGACTGGG 599 0.86 14.08 0.20 79.93 CCCAGUCGAG ATGGTCTCCGUCUGCCUCGC 364 AGTCAACTAC 600 0.96 3.53 0.13 86.86 GUAGUGACU GCGAGGCAGAAGGUAGUUCU 365 TTCCAGGATG 601 0.93 7.36 0.37 62.62 CAUCCUGGAA AGAACTACCTUCCUUGUAGU 366 AAGATCGTCC 602 0.87 12.96 0.15 84.87 GGACGAUCUUACTACAAGGA GCAUCCAAAA 367 CCAATCCACG 603 0.97 2.54 0.27 72.69 CGUGGAUUGGTTTTGGATGC GUCCAGCAGG 368 CCGACAACAT 604 0.70 30.00 0.17 82.64 AUGUGUCGGCCTGCTGGAC AGCUCCCGCA 369 GAGGTGACGC 605 0.86 13.72 0.20 80.40GCGUCACCUC TGCGGGAGCT CGAGAGCAGC 370 CCTCACTTGCG 606 1.02 -2.19 0.6337.11 GCAAGUGAGG CTGCTCTCG CAGGGAAAGC 371 TATCGGAGGC 607 0.89 11.10 0.0891.59 GCCUCCGAUA GCTTTCCCTG AUUUCAUAGG 372 GGTGTATTCG 608 1.05 -4.540.56 44.15 CGAAUACACC CCTATGAAAT UCGGCCAGGC 373 GGCCACATCC 609 0.7326.53 0.17 83.04 GGAUGUGGCC GCCTGGCCGA AAGGGAUGUG 374 GACTTCCGGA 6100.90 10.37 0.26 73.52 UCCGGAAGUC CACATCCCTT CUUGUAGUGG 375 GCAAGATCGT611 0.76 24.09 0.11 89.16 ACGAUCUUGC CCACTACAAG AGUCGGCCAG 376CCACATCCGC 612 0.94 6.15 0.33 67.44 GCGGAUGUGG CTGGCCGACT GCCUCAGCCU 377TCTTTCGGCCA 613 1.05 -4.82 0.37 63.11 GGCCGAAAGA GGCTGAGGC AGCGUCACCU378 GCTGAGGCCG 614 0.78 22.10 0.35 64.70 CGGCCUCAGC AGGTGACGCTCAGCGGCAGA 379 AGCACCTCTCT 615 0.96 4.49 0.14 86.00 GAGAGGUGCT CTGCCGCTGCCAGCGGCAG 380 GCACCTCTCTC 616 0.97 3.23 0.15 84.55 AGAGAGGUGC TGCCGCTGGUUGUAGUGGA 381 GGCAAGATCG 617 0.83 17.22 0.19 81.05 CGAUCUUGCCTCCACTACAA AGGGAAAGCG 382 CTATCGGAGG 618 1.01 -1.12 0.25 75.50CCUCCGAUAG CGCTTTCCCT GGGAAAGCGC 383 CCTATCGGAG 619 0.90 10.02 0.2376.79 CUCCGAUAGG GCGCTTTCCC

Example 8: Selected Antisense Oligonucleotides Provided Dose-DependentReduction in DMPK Expression in Immortalized Myoblasts

Eighteen oligonucleotides from Example 7 were selected to be evaluatedfor their ability to reduce DMPK expression in a dose-responsive manner.DM1 C15 myoblasts were prepared as in Example 7 to yield differentiatedmyotubes in 96-well microplates. After seven days of differentiation,cells were transfected with individual oligonucleotides usingLipofectamine MessengerMax. Each oligonucleotide was tested intriplicate at concentrations of 0.046 nM, 0.137 nM, 0.412 nM, 1.235 nM,3.704 nM, 11.11 nM, 33.33 nM, and 100 nM by 3-fold serial dilutionsusing 0.3 μL of Lipofectamine MessengerMax per well.

Following addition of oligonucleotide, cells were incubated for 72 hoursprior to harvesting for total RNA. cDNA was synthesized from the totalRNA extracts and qPCR was performed to determine expression levels ofDMPK using a commercially available Taqman probeset in technicalquadruplicate. All qPCR data were analyzed using a traditional ΔΔCTmethod and were normalized to a plate-based negative control thatcomprised of cells treated with vehicle control (0.3 μL/wellLipofectamine MessengerMax without any oligonucleotide). Data for eacholigonucleotide to was fit to sigmoidal curve in order to determine aneffective concentration of each oligonucleotide that provided ahalf-maximal response (EC-50). Results from these experiments are shownin Table 9.

Each of the eighteen antisense oligonucleotides selected fordose-dependent experimentation were capable of dose-dependently reducingDMPK in differentiated myotubes. Further, each of the tested antisenseoligonucleotides reduced DMPK with EC-50 values below 25 nM. Forexample, antisense oligonucleotides comprising SEQ ID NOs: 264, 215,222, 190, and 212 resulted in EC-50 values of 3.27 nM, 3.59 nM, 5.45 nM,6.04 nM, and 24.59 nM, respectively. These data demonstrate that theantisense oligonucleotides shown in Table 9 are capable ofdose-dependent reduction of DMPK in cellulo, suggesting thatmuscle-targeting complexes comprising these antisense oligonucleotideswould be capable of targeting DMPK in muscle tissues in vivo.

TABLE 9Ability ofDMPK-targeting antisense oligonucleotides to reduce expression of DMPKin dose-dependent manner in cellulo SEQ SEQ ResultsAntisense Oligonucleotide ID DMPK Target ID reduction at  Sequence NO:Sequence NO: EC-50 (nM) 100 nM GCAGGAUGUUGUCGGGU 201 CAAACCCGACAA 437 0.1679 89.77 UUG CATCCTGC AGCAGGAUGUUGUCGGG 218 AAACCCGACAAC 454 0.2266 85.81 UUU ATCCTGCT GCGUAGAAGGGCGUCUG 212 GGGCAGACGCCC 448 24.5995.13 CCC TTCTACGC CCCAGCGCCCACCAGUC 222 TGTGACTGGTGG 458  5.454 63.69ACA GCGCTGGG CCAUCUCGGCCGGAAUC 180 GCGGATTCCGGC 416  0.44 95.42 CGCCGAGATGG CGUUCCAUCUGCCCGCA 196 AGCTGCGGGCAG 432  0.19 89.97 GCU ATGGAACGCAGGGACAGCCGCUGGA 190 AGTTCCAGCGGC 426  6.04 90.59 ACU TGTCCCTGCAUGGCAUACACCUGGC 159 CGGGCCAGGTGT 395  0.42 75.28 CCG ATGCCATGGCUUCAUCUUCACUACC 188 AGCGGTAGTGAA 424  0.03 64.06 GCU GATGAAGCGAAUGUCCGACAGUGUC 195 GGAGACACTGTC 431  0.07 97.23 UCC GGACATTCGGACGAUCUUGCCAUAG 215 GACCTATGGCAA 451  3.59 92.18 GUC GATCGTCCGCUGUCCCGGAGACCAU 264 GGGATGGTCTCC 500  3.27 93.07 CCC GGGACAGCGACAGAACAACGGCGAA 248 CTGTTCGCCGTT 484  0.08 94.32 CAG GTTCTGTCUGUUGUCGGGUUUGAU 203 GGACATCAAACC 439  0.21 93.95 GUCC CGACAACACGAAUGUCCGACAGUGU 162 GAGACACTGTCG 398  0.18 95.93 CUC GACATTCGGGGCCUGGGACCUCACU 172 GACAGTGAGGTC 408  0.07 90.58 GUC CCAGGCCCCUCUGCCGCAGGGACAG 174 CGGCTGTCCCTG 410  0.42 93.66 CCG CGGCAGAGUUGCCAUAGGUCUCCGC 182 ACGGCGGAGACC 418  0.37 93.70 CGU TATGGCAA

Example 9: Targeting DMPK in Mouse Muscle Tissues with aMuscle-Targeting Complex

The RI7 217 Fab antibody-ASO muscle-targeting complex described inExample 2, DTX-C-008, was tested for time-dependent inhibition of DMPKin mouse tissues in vivo. C57BL/6 wild-type mice were intravenouslyinjected with a single dose of a vehicle control (phosphate-bufferedsaline (PBS)), naked antisense oligonucleotide (ASO300) (10 mg/kg ofASO), DTX-C-007 IgG2a Fab antibody-ASO control complex (10 mg/kg ofASO), or DTX-C-008 (10 mg/kg of ASO) on Day 0 and euthanized after aprescribed period of time, as described in Table 10. One group of micein each experimental condition was subjected to a second dose(multi-dose groups) at four weeks (Day 28). Following euthanization, themice were segmented into isolated tissue types and samples of tibialisanterior and gastrocnemius muscle tissues were subsequently assayed forexpression levels of DMPK (FIGS. 13A-13B).

TABLE 10 Experimental conditions Weeks after injection before NumberGroup Dosage euthanization of mice 1 Single dose of vehicle (PBS) 2 5 2Single dose of vehicle (PBS) 4 5 3 Single dose of vehicle (PBS) 8 3 4Multi-dose of vehicle (PBS) 8 2 5 Single dose of vehicle (PBS) 12 5 6Single dose of ASO300 2 5 7 Single dose of ASO300 4 5 8 Single dose ofASO300 8 5 9 Multi-dose of ASO300 8 5 10 Single dose of ASO300 12 5 11Single dose of DTX-C-007 control 2 5 12 Single dose of DTX-C-007 control4 5 13 Single dose of DTX-C-007 control 8 5 14 Multi-dose of DTX-C-007control 8 5 15 Single dose of DTX-C-007 control 12 5 16 Single dose ofDTX-C-008 2 5 17 Single dose of DTX-C-008 4 5 18 Single dose ofDTX-C-008 8 5 19 Multi-dose of DTX-C-008 8 5 20 Single dose of DTX-C-00812 5

Mice treated with the RI7 217 Fab antibody-ASO DTX-C-008 complexdemonstrated about 50-60% reduction in DMPK expression in tibialisanterior muscle (FIG. 13A) and about 30-50% reduction in DMPK expressionin gastrocnemius muscle (FIG. 13B) for all of Groups 16-20 (2-12 weeksbetween injection and euthanization), relative to vehicle. These datashow that a single dose of the muscle-targeting complex DTX-C-008reduces expression of DMPK for at least twelve weeks followingadministration of the complex.

In contrast, mice treated with the naked antisense oligonucleotide orthe control complex did not demonstrate significant inhibition of DMPKexpression across all experimental groups and tissues.

These data demonstrate that a muscle-targeting complex as describedherein is capable of providing persistent inhibition of DMPK expressionin vivo for up to 12 weeks following a single dose or administration ofsaid muscle targeting complex.

Example 10: A Muscle-Targeting Complex can Target Gene Expression in theNucleus

The RI7 217 Fab antibody-ASO muscle-targeting complex as described inExample 2, DTX-C-008, was tested for inhibition of nuclear-retained DMPKRNA in mouse muscle tissues. The mice used for this Example have beenengineered to express a human mutant DMPK gene (schematic shown in FIG.14A)—DMPK with a 350 CTG repeat region and a downstream G-for-Csingle-nucleotide polymorphism. As shown in FIG. 14A, the human mutantDMPK RNA is retained in the nucleus, while the mouse wild-type DMPK RNAis located in the cytoplasm and the nucleus.

Mice were intravenously injected with a single dose of a vehicle control(saline), an IgG2a Fab-ASO control complex DTX-C-007 (10 mg/kg of ASO),naked ASO300 (10 mg/kg of ASO), or DTX-C-008 (10 mg/kg of ASO) andeuthanized after 14 days. Six mice were treated in each experimentalcondition. Following euthanization, the mice were segmented intoisolated tissue types and tissue samples were subsequently assayed forexpression levels of mutant and wild-type DMPK (FIG. 14B).

Mice treated with the muscle-targeting RI7 217 Fab antibody-ASO complexDTX-C-008 demonstrated statistically significant reduction in bothnuclear-retained mutant DMPK and wild-type DMPK. (p-value<0.05). Thesedata demonstrate that a muscle-targeting complex as described herein iscapable of targeting DMPK in the nucleus.

Example 11: A Muscle-Targeting Complex Reverses Myotonia in HSA^(LR)Mouse Model

A muscle-targeting complex (DTX-Actin) was generated comprising anantisense oligonucleotide (ASO) that targets actin covalently linked toDTX-A-002 (RI7 217 Fab), an anti-transferrin receptor antibody.

The actin-targeting ASO is an MOE 5-10-5 gapmer that comprises:5′—NH₂—(CH₂)₆-dA*oC*oC*oA*oT*oT*dT*dT*dC*dT*dT*dC*dC*dA*dC*dA*oG*oG*oG*oC*oT-3(SEQ ID NO: 620); wherein ‘*’ represents a PS linkage; ‘d’ represents adeoxynucleic acid; and ‘o’ represents a 2′-MOE.

DTX-Actin was then tested for its ability to reduce target geneexpression (hACTA1) and reduce myotonia in HSA^(LR) mice, a mouse modelthat has a functional myotonia phenotype similar to that observed inhuman DM1 patients. Details of the HSA^(LR) mouse model are as describedin Mankodi, A. et al. Science. 289: 1769, 2000. HSA^(LR) mice wereintravenously injected with a single dose of PBS or DTX-Actin (either 10mg/kg or 20 mg/kg ASO equivalent). Each of these three experimentalconditions were replicated in two individual mice. On Day 14 afterinjection, mice were euthanized and specific muscles werecollected—quadriceps (quad), gastrocnemius (gastroc) and tibialisanterior (TA). The muscle tissues were analyzed for expression ofhACTA1. DTX-Actin demonstrated reduction of hACTA1 expression in allthree muscle tissues relative to vehicle control (FIG. 15A).

On Day 14 after injection, and prior to the euthanasia and tissuecollection described above, electromyography (EMG) was performed onspecific muscles. EMG myotonic discharges were graded by a blindedexaminer on a 4-point scale: 0, no myotonia; 1, occasional myotonicdischarge in less than 50% of needle insertions; 2, myotonic dischargein greater than 50% of needle insertions; and 3: myotonic discharge withnearly every insertion. DTX-Actin demonstrated reduction in gradedmyotonia in all three muscle tissues relative to vehicle control (FIG.15B). Mice treated with DTX-Actin at a dose of 20 mg/kg ASO equivalentdemonstrated little-to-no myotonia in quadriceps and gastrocnemiusmuscles.

These data demonstrate that a single dose of a muscle-targeting complexis capable of gene-specific targeting and reduction in functionalmyotonia in in the HSA^(LR) mice, a mouse model that has a functionalmyotonia phenotype similar to that observed in human DM1 patients.

Example 12: A Muscle-Targeting Complex can Functionally CorrectArrhythmia in a DM1 Mouse Model

The RI7 217 Fab antibody-ASO muscle-targeting complex as described inExample 2, DTX-C-008, was tested for its ability to functionally correctarrhythmia in a DM1 mouse model. The mice used for this Example are theoffspring of mice expressing myosin heavy chain reverse tettransactivator (MHCrtTA) and mice expressing a mutant form of human DMPK(CUG₉₆₀). FIG. 16A shows the genomic structure of the mutant transgene.

Doxycycline containing chow (2 g doxycycline/kg chow, Bio-Sere) wasprovided to the mice beginning at postnatal day 1, initially through thenursing dam and subsequently through chow after weaning, to induceselective expression of mutant DMPK in the heart. All mice weremaintained on chow containing doxycycline throughout the entire courseof the study except the “off Dox Control” group. At 12 weeks of age, allmice underwent a baseline pre-dose ECG evaluation. Mice were thentreated intravenously with a single dose of either vehicle (saline),naked ASO300 (10 mg/kg), DTX-C-008 (10 mg/kg ASO equivalent) orDTX-C-008 (20 mg/kg ASO equivalent). Following baseline pre-dose ECGevaluation mice in the “off Dox Control” group were switched to chowwithout doxycycline. Post dose ECG evaluations were performed in allmice 7 and 14 days after treatment, or in the case of the “off DoxControl” group 7 and 14 days after reversion to chow withoutdoxycycline. For each of the ECG spectra, QRS and QTc intervals weremeasured.

In this model, mice treated with doxycycline exhibit prolongation of QRSand QTC intervals driven by expression of mutant DMPK in the heart,similar to those reported in DM1 patients, and consistent with increasedpropensity for cardiac arrhythmia. Removal of doxycycline for the dietin the “Off Dox Control” group turns off expression of mutant DMPK,resulting in a normalization of QRS (FIG. 16B) and QTC (FIG. 16C)intervals. Mice maintained on doxycycline and treated with 20 mg/kg ofthe muscle-targeting complex DTX-C-008 demonstrated statisticallysignificant reduction in their QTc intervals after 14 days despitecontinued expression of mutant DMPK in the heart (FIG. 16C). Thisreduction in QTc intervals represents a correction in cardiac arrhythmiain a DM1 mouse model. These data demonstrate that a muscle-targetingcomplex as described herein is capable of providing a phenotypic andtherapeutic benefit in a DM1 model.

Example 13: A Muscle-Targeting Complex can Target DMPK and CorrectDM1-Related Genetic Splicing

In isolated muscles cells derived from human DM1 patients, themuscle-targeting complex as described in Example 5, DTX-C-012, wastested for reduction of DMPK expression and subsequent correction ofsplicing defects in Bin1, a downstream gene of DMPK.

Briefly, patient cells were seeded at a density of 10 k cells/wellbefore being allowed to recover overnight. Cells were then treated withPBS (vehicle control), naked ASO300, or DTX-C-012 (500 nM; equivalent to55.5 nM ASO). Cells were allowed to differentiate for 14 days.Expression levels of DMPK and % Bin1 exon-11 inclusion were determinedon Days 10, 11, 12, 13, and 14 post differentiation.

Treatment of DM1 patient cells with the DTX-C-012 complex leads toreduction of DMPK levels as early as Day 10 post differentiation (FIG.17A). Treatment of DM1 patient cells with the DTX-C-012 complex alsoleads to a statistically significant time-dependent change in Bin1splicing (FIG. 17B). (**p<0.01, ***p<0.001). These data demonstrate thata muscle-targeting complex as described herein is capable of providingphenotypic and therapeutic benefit (increased correction of DM1gene-specific splicing) in a DM1 model.

Example 14: Selected Antisense Oligonucleotides Provided Dose-DependentReduction in DMPK Expression in DM1 and NHP Myotubes

The antisense oligonucleotides listed in Table 9 were further assessedto identify oligos that are safe in vivo (e.g., as indicated by lowimmunogenicity as measured by cytokine induction), and further based onmanufacturability and secondary structure considerations. Threeantisense oligonucleotides from Table 9, (SEQ ID NO: 212, DMPK-ASO-1),(SEQ ID NO: 222, DMPK-ASO-2), and (SEQ ID NO: 180, DMPK-ASO-3) wereselected. These oligonucleotides were then further evaluated for theirability to reduce DMPK expression in DM1 myotubes and NHP myotubes in adose-responsive manner. Naked ASO300 was used as control. Each of theantisense oligonucleotides were capable of dose-dependently reducingDMPK in DM1 and NHP myotubes (see FIGS. 18A-18C and FIGS. 19A-19B,respectively).

These data demonstrate that these antisense oligonucleotides are safe invivo and are capable of dose-dependent reduction of DMPK in cellulo,suggesting that muscle-targeting complexes comprising these antisenseoligonucleotides would be capable of targeting DMPK in muscle tissues invivo.

Example 15: Binding Affinity of Selected Anti-TfR1 Antibodies to HumanTfR1

Selected anti-TfR1 antibodies were tested for their binding affinity tohuman TfR1 for measurement of Ka (association rate constant), Kd(dissociation rate constant), and K_(D) (affinity). Two known anti-TfR1antibodies were used as control, 15G11 and OKT9. The binding experimentwas performed on Carterra LSA at 25° C. An anti-mouse IgG and anti-humanIgG antibody “lawn” was prepared on a HC30M chip by amine coupling. 59IgGs (58 mouse mAbs and 1 human mAb) were captured on the chip. Dilutionseries of hTfR1, cyTfR1, and hTfR2 were injected to the chip for binding(starting from 1000 nM, 1:3 dilution, 8 concentrations).

Binding data were referenced by subtracting the responses from a bufferanalyte injection and globally fitting to a 1:1 Langmuir binding modelfor estimate of Ka (association rate constant), Kd (dissociation rateconstant), and K_(D) (affinity) using the Carterra™ Kinetics software.5-6 concentrations were used for curve fitting.

The result showed that the mouse mAbs demonstrated binding to hTfR1 withK_(D) values ranging from 13 μM to 50 nM. A majority of the mouse mAbshad K_(D) values in the single digit nanomolar to sub-nanomolar range.The tested mouse mAbs showed cross-reactive binding to cyTfR1 with K_(D)values ranging from 16 μM to 22 nM.

Ka, Kd, and K_(D) values of anti-TfR1 antibodies are provided in Table11.

TABLE 11 Ka, Kd, and K_(D) values of anti-TfR1 antibodies Name K_(D) (M)Ka (M) Kd (M) ctrl-15G11 2.83E−10 3.70E+05 1.04E−04 ctrl-OKT9 mIgG5.36E−10 7.74E+05 4.15E−04 3-A04 4.36E−10 4.47E+05 1.95E−04 3-M127.68E−10 1.66E+05 1.27E−04 5-H12 2.08E−07 6.67E+04 1.39E−02

Example 16: Conjugation of Anti-TfR1 Antibodies with Oligonucleotides

Complexes containing an anti-TfR1 antibody covalently conjugated toASO300 were generated. First, Fab fragments of anti-TfR antibody clones3-A4, 3-M12, and 5-H12 were prepared by cutting the mouse monoclonalantibodies with an enzyme in or below the hinge region of the full IgGfollowed by partial reduction. The Fabs were confirmed to be comparableto mAbs in avidity or affinity.

Muscle-targeting complexes was generated by covalently linking theanti-TfR mAbs to the ASO300 via a cathepsin cleavable linker. Briefly, aBicyclo[6.1.0]nonyne-PEG3-L-valine-L-citrulline-pentafluorophenyl ester(BCN-PEG3-Val-Cit-PFP) linker molecule was coupled to ASO300 through acarbamate bond. Excess linker and organic solvents were removed bytangential flow filtration (TFF). The purified Val-Cit-linker-ASO wasthen coupled to an azide functionalized anti-transferrin receptorantibody generated through modifying ε-amine on lysine withAzide-PEG4-PFP. A positive control muscle-targeting complex was alsogenerated using 15G11.

The product of the antibody coupling reaction was then subjected to twopurification methods to remove free antibody and free payload: 1)hydrophobic interaction chromatography (HIC-HPLC), and 2) Size exclusionchromatography (SEC). The HIC column utilized a decreasing salt gradientto separate free antibody from conjugate. During SEC, fractionation wasperformed based on A260/A280 traces to specifically collect conjugatedmaterial. Concentrations of the conjugates were determined by eitherNanodrop A280 or BCA protein assay (for antibody) and Quant-It Ribogreenassay (for payload). Corresponding drug-antibody ratios (DARs) werecalculated. DARs ranged between 0.8 and 2.0, and were standardized sothat all samples receive equal amounts of payload.

The purified complexes were then tested for cellular internalization andinhibition of the target gene, DMPK. Non-human primate (NHP) or DM1(donated by DM1 patients) cells were grown in 96-well plates anddifferentiated into myotubes for 7 days. Cells were then treated withescalating concentrations (0.5 nM, 5 nM, 50 nM) of each complex for 72hours. Cells were harvested, RNA was isolated, and reverse transcriptionwas performed to generate cDNA. qPCR was performed using TaqMan kitsspecific for Ppib (control) and DMPK on the QuantStudio 7. The relativeamounts of remaining DMPK transcript in treated vs non-treated cells waswere calculated and the results are shown in Table 12.

The results demonstrated that the anti-TfR1 antibodies are able totarget muscle cells, be internalized by the muscle cells with themolecular payload (ASO300), and that the molecular payload is able totarget and knockdown the target gene (DMPK).

TABLE 12 Binding Affinity of anti-TfR1 Antibodies and Efficacy ofConjugates % knockdown of % knockdown DMPK in cells of DMPK in fromhuman DM1 huTfR1 Avg cyTfR1 Avg NHP cells using patients using K_(D) (M)K_(D) (M) Antibody-DMPK Antibody-DMPK Clone Name (antibody alone)(antibody alone) ASO conjugate ASO conjugate 15G11 (control)  8.0E−10 1.0E−09 36 46 3-A4 4.36E−10 2.32E−09 77 70 3-M12 7.68E−10 5.18E−09 7752 5-H12 2.02316E−07   1.20E−08 88 57

Interestingly, the DMPK knockdown results showed a lack of correlationbetween the binding affinity of the anti-TfR to transferrin receptor andefficacy in delivering a DMPK-targeting ASO to cells for DMPK knockdown.Surprisingly, the anti-TfR antibodies provided herein (e.g., at least3-A4, 3-M12, and 5-H12) demonstrated superior activity in delivering apayload (e.g., DMPK ASO) to the target cells and achieving thebiological effect of the molecular payload (e.g., DMPK knockdown) ineither cyno cells or human DM1 patient cells, compared to the controlantibody 15G11, despite the comparable binding affinity (or, in certaininstances, such as 5-H12, lower binding affinity) to human or cynotransferrin receptor between these antibodies and the control antibody15G11.

Top attributes such as high huTfR1 affinity, >50% knockdown of DMPK inNHP and DM1 patient cell line, identified epitope binding with 3 uniquesequences, low/no predicted PTM sites, and good expression andconjugation efficiency led to the selection of the top 3 clones forhumanization, 3-A4, 3-M12, and 5-H12.

Example 17: Humanized Anti-TfR1 Antibodies

The anti-TfR antibodies shown in Table 2 were subjected to humanizationand mutagenesis to reduce manufacturability liabilities. The humanizedvariants were screened and tested for their binding properties andbiological actives. Humanized variants of anti-TfR1 heavy and lightchain variable regions (5 variants each) were designed using CompositeHuman Technology. Genes encoding Fabs having these heavy and light chainvariable regions were synthesized, and vectors were constructed toexpress each humanized heavy and light chain variant. Subsequently, eachvector was expressed on a small scale and the resultant humanizedanti-TfR1 Fabs were analyzed. Humanized Fabs were selected for furthertesting based upon several criteria including Biacore assays of antibodyaffinity for the target antigen, relative expression, percent homologyto human germline sequence, and the number of MHC class II predicted Tcell epitopes (determined using iTope™ MCH class II in silico analysis).

Potential liabilities were identified within the parental sequence ofsome antibodies by introducing amino acid substitutions in the heavychain and light chain variable regions. These substitutions were chosenbased on relative expression levels, iTope™ score and relative K_(D)from Biacore single cycle kinetics analysis. The humanized variants weretested and variants were selected initially based upon affinity for thetarget antigen. Subsequently, the selected humanized Fabs were furtherscreened based on a series of biophysical assessments of stability andsusceptibility to aggregation and degradation of each analyzed variant,shown in Table 14 and Table 15. The selected Fabs were analyzed fortheir properties binding to TfR1 by kinetic analysis. The results ofthese analyses are shown in Table 16. For conjugates shown in Table 14and Table 15, the selected humanized Fabs were conjugated to aDMPK-targeting oligonucleotide ASO300. The selected Fabs are thermallystable, as indicated by the comparable binding affinity to human andcyno TfR1 after been exposed to high temperature (40° C.) for 9 days,compared to before the exposure (see Table 16).

TABLE 14 Biophysical assessment data for humanized anti-TfR Fabs Variant3M12 3M12 3M12 3M12 3A4 (VH3- Criteria (VH3/Vk2) (VH3/Vk3) (VH4/Vk2)(VH4/Vk3) N54T/Vk4) Binding Affinity 395 pM 345 pM 396 pM 341 pM 3.09 nM(Biacore d0) Binding Affinity 567 pM 515 pM 510 pM 486 pM 3.01 nM(Biacore d25) Fab binding 0.8 nM/9.9 nM 0.6 nM/4.7 nM 0.4 nM/1.4 nM 0.5nM/2.2 nM 2.6 nM/156 nM* affinity ELISA (human/cyno TfR1) Conjugatebinding 2.2 nM/2.9 nM N/A N/A 1.7 nM/2.1 nM 2.8 nM/4.7 nM  affinityELISA (human/cyno TfR1) . . . Variant 3A4 (VH3- 3A4 5H12 (VH5- 5H12(VH5- 5H12 (VH4- Criteria N54S/Vk4) (VH3/Vk4) C33Y/Vk3) C33D/Vk4)C33Y/Vk4) Binding Affinity 1.34 nM 1.5 nM 627 pM 991 pM 626 pM (Biacored0) Binding Affinity 1.39 nM 1.35 nM 1.07 nM 3.01 nM 1.33 nM (Biacored25) Fab binding 1.6 nM/398 nM* 1.5 nM/122 nM*  6.3 nM/2.1 nM 6.0 nM/3.5nM   2.8 nM/3.3 nM affinity ELISA (human/cyno TfR1) Conjugate binding2.9 nM/7.8 nM  2.8 nM/7.6 nM  33.4 nM/2.3 nM 110 nM/10.2 nM 23.7 nM/3.3nM affinity ELISA (human/cyno TfR1) *Regains cyno binding afterconjugation;

TABLE 15 Thermal Stability for humanized anti-TfR Fabs and conjugatesVariant 3M12 3M12 3M12 3M12 3A4 (VH3- Criteria (VH3/Vk2) (VH3/Vk3)(VH4/Vk2) (VH4/Vk3) N54T/Vk4) Binding affinity 0.8 0.6 0.4 0.5 2.6 hTfR1d0 (nM) Binding affinity 0.98 1.49 0.50 0.28 0.40 hTfR1 d9 (nM) Bindingaffinity 9.9 4.7 1.4 2.2 156 cyno TfR1 d0 (nM) Binding affinity 19.5115.58 5.01 16.40 127.50 cyno TfR1 d9 (nM) DMPK oligo 1.14 N/A N/A 1.182.22 conjugate binding to hTfR1 (nM) DMPK oligo 2.26 N/A N/A 1.85 5.12conjugate binding to cyno TfR1 (nM) . . . Variant 3A4 (VH3- 3A4 5H12(VH5- 5H12 (VH5- 5H12 (VH4- Criteria N54S/Vk4) (VH3/Vk4) C33Y/Vk3)C33D/Vk4) C33Y/Vk4) Binding affinity 1.6 1.5 6.3 6 2.8 hTfR1 d0 (nM)Binding affinity 0.65 0.46 71.90 92.34 1731.00 hTfR1 d9 (nM) Bindingaffinity 398 122 2.1 3.5 3.3 cyno TfR1 d0 (nM) Binding affinity 248.30878.40 0.69 0.63 0.26 cyno TfR1 d9 (nM) DMPK oligo 2.71 2.837 N/A 110.513.9 conjugate binding to hTfR1 (nM) DMPK oligo 4.1 7.594 N/A 10.18 13.9conjugate binding to cyno TfR1 (nM)

TABLE 16 Kinetic analysis of humanized anti-TfR Fabs binding to TfR1Humanized anti-TfR Fabs k_(a) (1/Ms) k_(d) (1/s) K_(D) (M) R_(MAX) Chi²(RU²) 3A4 (VH3/Vk4) 7.65E+10 1.15E+02 1.50E−09 48.0 0.776 3A4(VH3-N54S/Vk4) 4.90E+10 6.56E+01 1.34E−09 49.4 0.622 3A4 (VH3-N54T/Vk4)2.28E+05 7.05E−04 3.09E−09 61.1 1.650 3M12 (VH3/Vk2) 2.64E+05 1.04E−043.95E−10 78.4 0.037 3M12 (VH3/Vk3) 2.42E+05 8.34E−05 3.45E−10 91.1 0.0253M12 (VH4/Vk2) 2.52E+05 9.98E−05 3.96E−10 74.8 0.024 3M12 (VH4/Vk3)2.52E+05 8.61E−05 3.41E−10 82.4 0.030 5H12 (VH5-C33D/Vk4) 6.78E+056.72E−04 9.91E−10 49.3 0.093 5H12 (VH5-C33Y/Vk3) 1.95E+05 1.22E−046.27E−10 68.5 0.021 5H12 (VH5-C33Y/Vk4) 1.86E+05 1.17E−04 6.26E−10 75.20.026Binding of Humanized Anti-TfR1 Fabs to TfR1 (ELISA)

To measure binding of humanized anti-TfR antibodies to TfR1, ELISAs wereconducted. High binding, black, flat bottom, 96 well plates (Corning#3925) were first coated with 100 μL/well of recombinant huTfR1 at 1μg/mL in PBS and incubated at 4° C. overnight. Wells were emptied andresidual liquid was removed. Blocking was conducted by adding 200 μL of1% BSA (w/w) in PBS to each well. Blocking was allowed to proceed for 2hours at room temperature on a shaker at 300 rpm. After blocking, liquidwas removed and wells were washed three times with 300 μL of TBST.Anti-TfR1 antibodies were then added in 0.5% BSA/TBST in triplicate inan 8 point serial dilution (dilution range 5 μg/mL— 5 ng/mL). A positivecontrol and isotype controls were also included on the ELISA plate. Theplate was incubated at room temperature on an orbital shaker for 60minutes at 300 rpm, and the plate was washed three times with 300 μL ofTBST. Anti-(H+L)IgG-A488 (1:500) (Invitrogen #A11013) was diluted in0.5% BSA in TBST, and 100 μL was added to each well. The plate was thenallowed to incubate at room temperature for 60 minutes at 300 rpm onorbital shaker. The liquid was removed and the plate was washed fourtimes with 300 μL of TBST. Absorbance was then measured at 495 nmexcitation and 50 nm emission (with a 15 nm bandwidth) on a platereader. Data was recorded and analyzed for EC₅₀. The data for binding tohuman TfR1 (hTfR1) for the humanized 3M12, 3A4 and 5H12 Fabs are shownin FIGS. 21A, 21C, and 21E, respectively. ELISA measurements wereconducted using cynomolgus monkey (Macaca fascicularis) TfR1 (cTfR1)according to the same protocol described above for hTfR1, and resultsare shown in FIGS. 21B, 21D, and 21F.

Results of these two sets of ELISA analyses for binding of the humanizedanti-TfR Fabs to hTfR1 and cTfR1 demonstrate that humanized 3M12 Fabsshow consistent binding to both hTfR1 and cTfR1, and that humanized 3A4Fabs show decreased binding to cTfR1 relative to hTfR1.

Antibody-oligonucleotide conjugates were prepared using six humanizedanti-TfR Fabs, each of which were conjugated to a DMPK targetingoligonucleotide ASO300. Conjugation efficiency and down-streampurification were characterized, and various properties of the productconjugates were measured. The results demonstrate that conjugationefficiency was robust across all 10 variants tested, and that thepurification process (hydrophobic interaction chromatography followed byhydroxyapatite resin chromatography) were effective. The purifiedconjugates showed a >97% purity as analyzed by size exclusionchromatography.

Several humanized Fabs were tested in cellular uptake experiments toevaluate TfR1-mediated internalization. To measure such cellular uptakemediated by antibodies, humanized anti-TfR Fab conjugates were labeledwith Cypher5e, a pH-sensitive dye. Rhabdomyosarcoma (RD) cells weretreated for 4 hours with 100 nM of the conjugates, trypsinized, washedtwice, and analyzed by flow cytometry. Mean Cypher5e fluorescence(representing uptake) was calculated using Attune N×T software. As shownin FIG. 22 , the humanized anti-TfR Fabs show similar or greaterendosomal uptake compared to a positive control anti-TfR1 Fab. Similarinternalization efficiencies were observed for different oligonucleotidepayloads. An anti-mouse TfR antibody was used as the negative control.Cold (non-internalizing) conditions abrogated the fluorescence signal ofthe positive control antibody-conjugate (data not shown), indicatingthat the positive signal in the positive control and humanized anti-TfRFab-conjugates is due to internalization of the Fab-conjugates.

Conjugates of six humanized anti-TfR Fabs of were also tested forbinding to hTfR1 and cTfR1 by ELISA, and compared to the unconjugatedforms of the humanized Fabs. Results demonstrate that humanized 3M12 and5H12 Fabs maintain similar levels of hTfR1 and cTfR1 binding afterconjugation relative to their unconjugated forms (3M12, FIGS. 23A and23B; 5H12, FIGS. 23E and 23F). Interestingly, 3A4 clones show improvedbinding to cTfR1 after conjugation relative to their unconjugated forms(FIGS. 23C and 23D).

As used in this Example, the term ‘unconjugated’ indicates that theantibody was not conjugated to an oligonucleotide.

Example 18. Knockdown of DMPK mRNA Level Facilitated by OligonucleotidesConjugates In Vitro

DMPK-targeting oligonucleotides (e.g., ASO) were tested inrhabdomyosarcoma (RD) cells for knockdown of DMPK transcript expression.RD cells were cultured in a growth medium of DMEM with glutamine,supplemented with 10% FBS and penicillin/streptomycin until nearlyconfluent. Cells were then seeded into a 96 well plate at 20K cells perwell and were allowed to recover for 24 hours. Cells were then treatedwith free DMPK-targeting oligonucleotides or by transfection of theoligonucleotides using 0.3 μL per well of Lipofectamine MessengerMAXtransfection reagent. After 3 days, total RNA was collected from cells,cDNA was synthesized and DMPK expression was measured by qPCR.

Results in FIG. 24 show that DMPK expression level was reduced in cellstreated with each given DMPK-targeting oligonucleotide, relative toexpression in PBS-treated cells. Several DMPK-oligonucleotides showeddose-dependent reduction of DMPK expression level. In FIG. 24 ,DMPK-ASO-1 has the sequence GCGUAGAAGGGCGUCUGCCC (SEQ ID NO: 212).DMPK-ASO-2 has the sequence CCCAGCGCCCACCAGUCACA (SEQ ID NO: 222).DMPK-ASO-3 has the sequence CCAUCUCGGCCGGAAUCCGC (SEQ ID NO: 180).ASO300 was also used in this experiment.

Example 19. Knockdown of DMPK mRNA Level Facilitated byAntibody-Oligonucleotide Conjugates In Vitro

Conjugates containing humanized anti-TfR Fabs 3M12(VH3/Vk2), 3M-12(VH4/Vk3), and 3A4(VH3-N54S/Vk4) were conjugated to a DMPK-targetingoligonucleotide (ASO300) and were tested in rhabdomyosarcoma (RD) cellsfor knockdown of DMPK transcript expression. Antibodies were conjugatedto ASO300 via the linker shown in Formula (C).

RD cells were cultured in a growth medium of DMEM with glutamine,supplemented with 10% FBS and penicillin/streptomycin until nearlyconfluent. Cells were then seeded into a 96 well plate at 20K cells perwell and were allowed to recover for 24 hours. Cells were then treatedwith the conjugates for 3 days. Total RNA was collected from cells, cDNAwas synthesized and DMPK expression was measured by qPCR.

Results in FIG. 25 show that DMPK expression level was reduced in cellstreated with each indicated conjugate, relative to expression inPBS-treated cells, indicating that the humanized anti-TfR Fabs are ableto mediate the uptake of the DMPK-targeting oligonucleotide by the RDcells and that the internalized DMPK-targeting oligonucleotide areeffective in knocking down DMPK mRNA level.

Example 20. Splicing Correction and Functional Efficacy in HSA-LR MouseModel of DM1

Correction of splicing in the HSA-LR mouse model of DM1 was demonstratedwith conjugates containing anti-TfR antibodies conjugated tooligonucleotides target human skeletal actin (ACTA1). The anti-TfR1antibody used in this study was RI7 217. The oligonucleotide targetingACTA1 is an MOE 5-10-5 gapmer that comprises:5′—NH₂—(CH₂)₆-dA*oC*oC*oA*oT*oT*dT*dT*dC*dT*dT*dC*dC*dA*dC*dA*oG*oG*oG*oC*oT-3(SEQ ID NO: 620); wherein ‘*’ represents a PS linkage; ‘d’ represents adeoxynucleic acid; and ‘o’ represents a 2′-MOE.

The HSA-LR DM1 mouse model is a well-validated model of DM1 thatexhibits pathologies that are very similar to human DM1 patients. TheHSA-LR model uses the human skeletal actin (ACTA1) promoter to driveexpression of CUG long repeats (LR). In this model, toxic DMPK RNAaccumulates within the nucleus and sequesters proteins responsible forsplicing, such as Muscleblind-like protein (MBNL), resulting inmis-splicing of multiple RNAs, including CLCN1 (chloride channel),ATP2a1 (calcium channel), and others. This mis-splicing causes the miceto also exhibit myotonia which is a hallmark of the DM1 clinicalpresentation in humans.

The anti-TfR-oligonucleotide conjugate delivered intravenously has beenshown to have activity in dose-dependent correction of splicing inmultiple RNAs and multiple muscles and was well tolerated by HSA-LRmice. In this study, the ability of the conjugates to correct splicingin more than 30 different RNAs was evaluated. In DM1, significant RNAmis-splicing of these RNAs reduces the ability of multiple muscles tofunction. The RNAs monitored are critical for contraction and relaxationof muscle in HSA-LR mice. Dose-dependent correction of splicing wasobserved.

FIG. 26 shows results for Atp2al, which encodes a calcium channel andcontributes to muscle contraction and relaxation. The X-axis representssplice derangement with 1.00 representing severe mis-splicing and 0.00representing a wild type (WT) splice pattern. Progression from right toleft in the figure represents a correction of splicing. The Y-axisrepresents the percent of the gene spliced in (PSI). Severe mis-splicingof ATP2a1 is caused by exclusion of exon 22 in the ATP2a1 RNA. WTsplicing reflects near complete inclusion of exon 22. Resultsdemonstrate that the conjugate corrected splicing of ATP2a1 in adose-dependent manner in the gastrocnemius muscle.

Data for the more than 30 different RNAs that were tested in this studyare shown in FIGS. 27, 35A, and 35B. Similar dose-dependent correctionof splicing was achieved for all of the tested RNAs in gastrocnemiusmuscle (FIG. 27 ), tibialis anterior (FIG. 35A), and quadriceps (FIG.35B). For some of these RNAs, correction of splicing is reflected by adecrease in PSI, as in FIG. 26 , and for other RNAs correction isreflected by an increase in PSI.

Similar dose-dependent improvements in splicing within the set of RNAswere observed in the quadriceps and tibialis anterior muscles, aftertreatment with the conjugate. FIG. 28 shows composite levels of splicingderangement observed for saline and different doses of the Ab-ASO acrossthe more than 30 RNAs that were tested in each muscle type. Doses of 10mg/kg and 20 mg/kg were administered in this study.

In addition to reductions in splicing derangement across multiple genesin several muscles in the HSA-LR model, disease modification wasobserved in the HSA-LR model. The results in FIG. 29 show that almostcomplete reversal of myotonia was achieved after a single dose of theconjugate. The severity of myotonia on a four-point scale was evaluated14 days following dosing with saline (PBS), naked oligonucleotide, orthe conjugate. Grade 0 indicates no myotonia was observed, grade 1indicates myotonic discharge was measured by electromyography (EMG) inless than 50% of needle insertions, grade 2 indicates myotonic dischargewas measured in greater than 50% of needle insertions and grade 3indicates myotonic discharge was measured with nearly every needleinsertion.

Example 21. DMPK-Targeting PMOs

Additional DMPK targeting oligonucleotides (PMOs) were designed andtested for their activity in reducing DMPK expression in primary humanmyotubes. Wild-type primary myoblasts were cultured in PromoCellSkeletal Muscle growth medium with 5% FBS and penicillin/streptomycinuntil nearly confluent. Cells were then seeded into a 96 well plate at50K cells per well and allowed to recover for 24 hours. Cells were thendifferentiated in a differentiation medium of DMEM with glutamine andpenicillin/streptomycin for 7 days. Cells were then treated with theunconjugated PMO for 3 days. Total RNA was collected from cells, cDNAwas synthesized and DMPK expression was measured by qPCR. The sequencesof the PMOs and their activity in knocking down DMPK in vitro are shownin Table 17.

As used in this Example, the term ‘unconjugated’ indicates that theoligonucleotide was not conjugated to an antibody.”

TABLE 17 DMPK-targeting PMOs and activity in knocking down DMPK in vitro% DMPK Knockdown in SEQ ID Primary Human PMO Sequence NO Target regionMyotubes CAGGTGACAGTTCAGGTGCAG 624 Intron 1-2 59% TCCACCCTGACTCCAGGTGAC625 Intron 1-2 16% GAGAAGGAAATAAGACCCAGTT 626 Intron 1-2 14%CCTTCTCTCTGCCTCTCAGCTT 627 Intron 1-2 58% CCACCCTCTGTCTGTCTCC 628Intron 1-2 64% TCCGCTGGGTGGTGGGAAAAGAA 629 Exon 2  9%ATGGGCTCCGCTGGGTGGTGG 630 Exon 2 11% ACGATGGGCTCCGCTGGG 631 Exon 2 60%CCATCCTTGGGCAGAGACCT 632 Intron 4-5 41% ATGACCAGGTACTGAGAAGGG 633 Exon 533% AGGTACTGAGAAGGGTTCGTC 634 Exon 5 40% TAGGGACCTGCGGAGAGGGCGA 635Exon 15 35% GCCTAGGGACCTGCGGGAGAG 636 Exon 15 62% GCCTTTTATTCGCGAGGGTCGG637 polyA 73% TGGAGGGCCTTTTATTCGCGAGG 638 polyA 66%TAGGCACTCACCCACTGCAAGA 621 Exon 1 69% CGGAGCTCACCAGGTAGTTCT 622Intron 4-5 73% AGGGCAGTGCTTACCTGAGGG 623 Intron 9-10 57%

Example 22. Serum Stability of the Linker Linking the Anti-TfR Antibodyand the Molecular Payload

Oligonucleotides which were linked to antibodies in examples wereconjugated via a cleavable linker shown in Formula (C). It is importantthat the linker maintain stability in serum and provide release kineticsthat favor sufficient payload accumulation in the targeted muscle cell.This serum stability is important for systemic intravenousadministration, stability of the conjugated oligonucleotide in thebloodstream, delivery to muscle tissue and internalization of thetherapeutic payload in the muscle cells. The linker has been confirmedto facilitate precise conjugation of multiple types of payloads(including ASOs, siRNAs and PMOs) to Fabs. This flexibility enabledrational selection of the appropriate type of payload to address thegenetic basis of each muscle disease. Additionally, the linker andconjugation chemistry allowed the optimization of the ratio of payloadmolecules attached to each Fab for each type of payload, and enabledrapid design, production and screening of molecules to enable use invarious muscle disease applications.

FIG. 20 shows serum stability of the linker in vivo, which wascomparable across multiple species over the course of 72 hours afterintravenous dosing. At least 75% stability was measured in each case at72 hours after dosing.

Example 23. In Vivo Activity of Anti-TfR Conjugates in hTfR1 Mice

In DM1, the higher than normal number of CUG repeats form large hairpinloops that remain trapped in the nucleus, forming nuclear foci that bindsplicing proteins and inhibit the ability of splicing proteins toperform their normal function. When toxic nuclear DMPK levels arereduced, the nuclear foci are diminished, releasing splicing proteins,allowing restoration of normal mRNA processing, and potentially stoppingor reversing disease progression.

The in vivo activity of conjugates containing an anti-TfR Fab (control,3M12 VH3/VK2, 3M12 VH4/VK3, 3A4 VH3 N54S/VK4) conjugated to theDMPK-targeting oligonucleotide (ASO300) in reducing DMPK mRNA level inmultiple muscle tissues following systemic intravenous administration inmice was evaluated.

Male and female C57BL/6 mice where one TfR1 allele was replaced with ahuman TFR1 allele were administered between the ages of 5 and 15 weeksaccording to the dosing schedule outlined in Table 18 and in FIG. 30A.Mice were sacrificed 14 days after the first injection and selectedmuscles collected as indicated in Table 19.

TABLE 18 Animal Treatment Treatment Dose Level Dose Volume DosingTerminal Group No. Antibody Oligo (mg/kg) (mL/kg) Regimen Time Point 1 4Vehicle NA 0 10 Day 0 and Day 14 2 4 NA ASO300 10 5.0 Day 7 by 3 4control ASO300 10.2 IV anti-TfR Fab 4 4 3M12 ASO300 11.5 VH3/VK2 5 43M12 ASO300 10.1 VH4/VK3 6 4 3A4 VH3 ASO300 10.7 N54S/VK4

TABLE 19 Tissue Storage Gastrocnemius Right leg of each animal stored inRNALater at −80° C. Tibialis One leg (R) of each animal stored Anteriorin RNALater at −80° C. Heart Dissect transversally and store the apex inRNAlater at −80° C. Diaphragm Split in half and collect one half inRNAlater at −80° C.

Total RNA was extracted on a Maxwell Rapid Sample Concentrator (RSC)Instrument using kits provided by the manufacturer (Promega). PurifiedRNA was reverse-transcribed and levels of Dmpk and Ppib transcriptsdetermined by qRT-PCR with specific TaqMan assays (ThermoFlsher). Logfold changes in Dmpk expression were calculated according to the2^(−ΔΔCT) method using Ppib as the reference gene and mice injected withvehicle as the control group. Statistical significance in differences ofDmpk expression between control mice and mice administered with theconjugates were determined by one-way ANOVA with Dunnet's correction formultiple comparisons. As shown in FIGS. 30B-30E, the tested conjugatesshowed robust activity in reducing DMPK mRNA level in vivo in variousmuscle tissues.

Example 24. In Vitro Activity of Anti-TfR Conjugates in Patient-DerivedCells

An in vitro experiment was conducted to determine the activities ofanti-TfR conjugates in reducing DMPK mRNA expression, correcting BIN1splicing, and reducing nuclear foci in CM-DM1-32F primary cellsexpressing a mutant DMPK mRNA containing 380 CUG repeats. The CM-DM1-32Fprimary cell is an immortalized myoblastic cell line isolated from a DM1patient (CL5 cells; Described in Arandel et al., Dis Model Mech. 2017Apr. 1; 10(4): 487-497). Conjugate 1 contains an anti-TfR mAb conjugatedto DMPK-targeting oligonucleotide (ASO300). Conjugate 2 contains ananti-TfR Fab conjugated to DMPK ASO-1 (GCGUAGAAGGGCGUCUGCCC; SEQ ID NO:212).

CL5 cells were seeded at a density of 156,000 cells/cm², allowed torecover for 24 hours, transferred to differentiation media to inducemyotube formation, as described (Arandel et al. Dis Model Mech. (2017)10(4):487-497) and subsequently exposed to conjugate 1 and conjugate 2at a payload concentration of 500 nM. Parallel cultures exposed tovehicle PBS served as controls. Cells were harvested after 10 days ofculture.

For analysis of gene expression, cells were collected with Qiazol fortotal RNA extraction with a Qiagen miRNAeasy kit. Purified RNA wasreverse-transcribed and levels of DMPK, PPIB, BIN1 transcripts and ofthe BIN1 mRNA isoform containing exon 11 determined by qRT-PCR withspecific TaqMan assays (ThermoFlsher). Log fold changes in DMPKexpression were calculated according to the 2^(−ΔΔCT) method using PPIBas the reference gene and cells exposed to vehicle as the control group.Log fold changes in the levels BIN1 isoform containing exon 11 werecalculated according to the 2^(−ΔΔCT) method using BIN1 as the referencegene and cells exposed to vehicle as the control group.

To measure the area of mutant DMPK nuclear foci, cells were fixed in 4%formalin, permeabilized with 0.1% Triton X-100 and hybridized at 70° C.with a CAG peptide-nucleic acid probe conjugated to the Cy5 fluorophore.After multiple washes in hybridization buffer and 2×SSC solution, nucleiwere counterstained with DAPI. Images were collected at a 400×magnification by confocal microscopy and foci area measured as the areaof Cy5 signal contained within the area of DAPI signal. Data wereexpressed as foci area corrected for nuclear area.

The results show that a single dose of the conjugates containing ananti-TfR (IgG or Fab) conjugated to a DMPK-targeting oligonucleotide(ASO300 or DMPK ASO-1 (SEQ ID NO: 212)) resulted reduced mutant DMPKexpression (FIG. 31A), corrected BIN1 splicing (FIG. 31B), and reducednuclear foci by approximately 40% (FIG. 31C).

Example 25. Characterization of Binding Activities of Anti-TfR Fab 3M12VH4/Vk3

In vitro studies were performed to test the specificity of anti-TfR Fab3M12 VH4/Vk3 for human and cynomolgus monkey TfR1 binding and to confirmits selectivity for human TfR1 vs TfR2. The binding affinity of anti-TfRFab 3M12 VH4/Vk3 to TfR1 from various species was determined using anenzyme-linked immunosorbent assay (ELISA). Serial dilutions of the Fabwere added to plates precoated with recombinant human, cynomolgusmonkey, mouse, or rat TfR1. After a short incubation, binding of the Fabwas quantified by addition of a fluorescently tagged anti-(H+L) IgGsecondary antibody and measurement of fluorescence intensity at 495 nmexcitation and 520 nm emission. The Fab showed strong binding affinityto human and cynomolgus monkey TfR1, and no detectable binding of mouseor rat TfR1 was observed (FIG. 32 ). Surface plasmon resonance (SPR)measurements were also conducted, and results are shown in Table 20. TheKd of the Fab against the human TfR1 receptor was calculated to be7.68×10⁻¹° M and against the cynomolgus monkey TfR1 receptor wascalculated to be 5.18×10⁻⁹M.

TABLE 20 Kinetic analysis of anti-TfR Fab 3M12 VH4/Vk3 binding to humanand cynomolgus monkey TfR1 or human TfR2, measured using surface plasmonresonance Anti-TfR Fab 3M12 VH4/Vk3 K_(d) k_(a) k_(d) R_(es) Target (M)(M⁻¹ s⁻¹) (s⁻¹) R_(max) SD Human TfR1 7.68E−10 1.66E+05 1.27E−041.11E+02 3.45E+00 Cyno TfR1 5.18E−09 9.19E+04 4.76E−04 1.87E+02 6.24E+00Human TfR2 ND ND ND ND ND ND = No detectable binding by SPR (10 pM-100uM)

To test for cross-reactivity of anti-TfR Fab 3M12 VH4/Vk3 to human TfR2,an ELISA was performed. Recombinant human TfR2 protein was platedovernight at 2 μg/mL and was blocked for 1 hour with 1% bovine serumalbumin (BSA) in PBS. Serial dilutions of the Fab or a positive controlanti-TfR2 antibody were added in 0.5% BSA/TBST for 1 hour. Afterwashing, anti-(H+L) IgG-A488 (Invitrogen #MA5-25932) fluorescentsecondary antibody was added at a 1:500 dilution in 0.5% BSA/TBST andthe plate was incubated for 1 hour. Relative fluorescence was measuredusing a Biotek Synergy plate reader at 495 nm excitation and 520 nmemission. No binding of anti-TfR Fab 3M12 VH4/Vk3 to hTfR2 was observed(FIG. 33 ).

Example 26. Serum Stability of Anti-TfR Fab-ASO Conjugate

Anti-TfR Fab VH4/Vk3 was conjugated to a control antisenseoligonucleotide (ASO) via a linker as shown in Formula (C) and theresulting conjugate was tested for stability of the linker conjugatingthe Fab to the ASO. Serum stability was measured by incubatingfluorescently labeled conjugate in PBS or in rat, mouse, cynomolgusmonkey, or human serum and measuring relative fluorescence intensityover time, with higher fluorescence indicating more conjugate remainingintact. FIG. 34 shows serum stability was similar across multiplespecies and remained high after 72 hours.

Example 27. Effect of a Single Dose of an Anti-Mouse TfR Fab Conjugatedto an Oligonucleotide (ASO) Against the Human ACTA1 mRNA in HSA^(LR)Mice, a Model of DM1

Six- to 8-week-old homozygous HSA^(LR) mice were allocated randomly toone of five treatment groups and treated with vehicle, a nakedoligonucleotide (ASO) targeting human ACTA1 mRNA at a dose of 10 mg/kgor 20 mg/kg, or conjugates containing anti-TfR RI7217 Fab conjugated tothe ASO (Ab-ASO) at a dose equivalent to 10 mg/kg or 20 mg/kg of ASO.

Twenty-eight days after intravenous injections of vehicle, ASO, orAb-ASO, EMG myotonic discharges in quadriceps (FIG. 36A), gastrocnemius(FIG. 36B), and tibialis anterior (FIG. 36C), were graded by blindedexaminer on a 4-point scale in which 0 indicated no myotonia; 1indicated occasional myotonic discharge in less than 50% of needleinsertions; 2 indicated myotonic discharge in greater than 50% of needleinsertions; and 3 indicated myotonic discharge with nearly everyinsertion. A single dose of the conjugate, but not naked ASO,dose-dependently reversed myotonia in the HSA^(LR) DM1 model.

Statistical significance of differences between vehicle-treated groupand each experimental arm was determined by Kruskal-Wallis test withDunn's multiple comparisons test. Data are reported as means±S.E.M.;*p<0.05, **p<0.01.

Additionally, fourteen days and twenty-eight days after the treatmentmice were sacrificed and quadriceps (quad), gastrocnemius (gastroc) andtibialis anterior (TA) muscles were collected and analyzed forexpression of ACTA1. Knockdown (KD) of ACTA1 expression using the Ab-ASOconjugate was observed in quadriceps (quad), gastrocnemius (gastroc) andtibialis anterior (TA) muscles relative to PBS control 14 days followinga single administered dose, whereas naked ASO did not facilitate ACTA1suppression in the same timeframe (FIG. 37 ). Measurement of ACTA1 inmuscle 28 days following the treatment showed that the, the Ab-ASOconjugate reduced ACTA1 expression relative to vehicle control at bothtested doses (10 mg/kg ASO equivalent and 20 mg/kg ASO equivalent),whereas the same doses of naked ASO did not facilitate ACTA1 suppression(FIGS. 38A-38C; *p<0.05, **p <0.01).

ADDITIONAL EMBODIMENTS

1. A complex comprising a muscle-targeting agent covalently linked to amolecular payload configured for inhibiting expression or activity of aDMPK allele comprising a disease-associated-repeat, wherein themuscle-targeting agent specifically binds to an internalizing cellsurface receptor on muscle cells,

wherein the muscle targeting agent is a humanized antibody that binds toa transferrin receptor, wherein the antibody comprises:

(i) a heavy chain variable region (VH) comprising an amino acid sequenceat least 85% identical to SEQ ID NO: 69; and/or a light chain variableregion (VL) comprising an amino acid sequence at least 85% identical toSEQ ID NO: 70;

(ii) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 71; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 70;

(iii) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 72; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 70;

(iv) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 73; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 74;

(v) a heavy chain variable region (VH) comprising an amino acid sequenceat least 85% identical to SEQ ID NO: 73; and/or a light chain variableregion (VL) comprising an amino acid sequence at least 85% identical toSEQ ID NO: 75;

(vi) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 76; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 74;

(vii) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 76; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 75;

(viii) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 77; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 78;

(ix) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 79; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 80; or

(x) a heavy chain variable region (VH) comprising an amino acid sequenceat least 85% identical to SEQ ID NO: 77; and/or a light chain variableregion (VL) comprising an amino acid sequence at least 85% identical toSEQ ID NO: 80.

2. The complex of embodiment 1, wherein the antibody comprises:

(i) a VH comprising the amino acid sequence of SEQ ID NO: 69 and a VLcomprising the amino acid sequence of SEQ ID NO: 70;

(ii) a VH comprising the amino acid sequence of SEQ ID NO: 71 and a VLcomprising the amino acid sequence of SEQ ID NO: 70;

(iii) a VH comprising the amino acid sequence of SEQ ID NO: 72 and a VLcomprising the amino acid sequence of SEQ ID NO: 70;

(iv) a VH comprising the amino acid sequence of SEQ ID NO: 73 and a VLcomprising the amino acid sequence of SEQ ID NO: 74;

(v) a VH comprising the amino acid sequence of SEQ ID NO: 73 and a VLcomprising the amino acid sequence of SEQ ID NO: 75;

(vi) a VH comprising the amino acid sequence of SEQ ID NO: 76 and a VLcomprising the amino acid sequence of SEQ ID NO: 74;

(vii) a VH comprising the amino acid sequence of SEQ ID NO: 76 and a VLcomprising the amino acid sequence of SEQ ID NO: 75;

(viii) a VH comprising the amino acid sequence of SEQ ID NO: 77 and a VLcomprising the amino acid sequence of SEQ ID NO: 78;

(ix) a VH comprising the amino acid sequence of SEQ ID NO: 79 and a VLcomprising the amino acid sequence of SEQ ID NO: 80; or

(x) a VH comprising the amino acid sequence of SEQ ID NO: 77 and a VLcomprising the amino acid sequence of SEQ ID NO: 80.

3. The complex of embodiment 1 or embodiment 2, wherein the antibody isselected from the group consisting of a full-length IgG, a Fab fragment,a Fab′ fragment, a F(ab′)2 fragment, a scFv, and a Fv.

4. The complex of embodiment 3, wherein the antibody is a full-lengthIgG, optionally wherein the full-length IgG comprises a heavy chainconstant region of the isotype IgG1, IgG2, IgG3, or IgG4.

5. The complex of embodiment 4, wherein the antibody comprises:

(i) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 84; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 85;

(ii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 86; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 85;

(iii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 87; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 85;

(iv) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 88; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 89;

(v) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 88; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 90;

(vi) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 91; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 89;

(vii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 91; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 90;

(viii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 92; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 93;

(ix) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 94; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 95; or

(x) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 92; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 95.

6. The complex of embodiment 3, wherein the antibody is a Fab fragment.

7. The complex of embodiment 6, wherein the antibody comprises:

(i) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 97; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 85;

(ii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 98; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 85;

(iii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 99; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 85;

(iv) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 100; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 89;

(v) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 100; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 90;

(vi) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 101; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 89;

(vii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 101; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 90;

(viii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 102; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 93;

(ix) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 103; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 95; or

(x) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 102; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 95.

8. The complex of embodiment 6 or embodiment 7, wherein the antibodycomprises:

(i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 97;and a light chain comprising the amino acid sequence of SEQ ID NO: 85;

(ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 98;and a light chain comprising the amino acid sequence of SEQ ID NO: 85;

(iii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 99;and a light chain comprising the amino acid sequence of SEQ ID NO: 85;

(iv) a heavy chain comprising the amino acid sequence of SEQ ID NO: 100;and a light chain comprising the amino acid sequence of SEQ ID NO: 89;

(v) a heavy chain comprising the amino acid sequence of SEQ ID NO: 100;and a light chain comprising the amino acid sequence of SEQ ID NO: 90;

(vi) a heavy chain comprising the amino acid sequence of SEQ ID NO: 101;and a light chain comprising the amino acid sequence of SEQ ID NO: 89;

(vii) a heavy chain comprising the amino acid sequence of SEQ ID NO:101; and a light chain comprising the amino acid sequence of SEQ ID NO:90;

(viii) a heavy chain comprising the amino acid sequence of SEQ ID NO:102; and a light chain comprising the amino acid sequence of SEQ ID NO:93;

(ix) a heavy chain comprising the amino acid sequence of SEQ ID NO: 103;and a light chain comprising the amino acid sequence of SEQ ID NO: 95;or

(x) a heavy chain comprising the amino acid sequence of SEQ ID NO: 102;and a light chain comprising the amino acid sequence of SEQ ID NO: 95.

9. The complex of any one of embodiments 1 to 8, wherein the equilibriumdissociation constant (K_(D)) of binding of the antibody to thetransferrin receptor is in a range from 10⁻¹¹ M to 10⁻⁶ M.

10. The complex of any one of embodiments 1 to 9, wherein the antibodydoes not specifically bind to the transferrin binding site of thetransferrin receptor and/or wherein the antibody does not inhibitbinding of transferrin to the transferrin receptor.

11. The complex of any one of embodiments 1 to 10, wherein the antibodyis cross-reactive with extracellular epitopes of two or more of a human,non-human primate and rodent transferrin receptor.

12. The complex of any one of embodiments 1 to 11, wherein the complexis configured to promote transferrin receptor mediated internalizationof the molecular payload into a muscle cell.

13. The complex of any one of embodiments 1 to 12, wherein the antibodyis a chimeric antibody, optionally wherein the chimeric antibody is ahumanized monoclonal antibody.

14. The complex of any one of embodiments 1 to 13, wherein the antibodyis in the form of a ScFv, Fab fragment, Fab′ fragment, F(ab′)₂ fragment,or Fv fragment. 15. The complex of any one of embodiments 1 to 14,wherein the molecular payload is an oligonucleotide.

16. The complex of embodiment 15, wherein the oligonucleotide comprisesat least 15 consecutive nucleotides of a sequence comprising any one ofSEQ ID NOs: 148-383 and 621-638.

17. The complex of embodiment 16, wherein the oligonucleotide comprisesa sequence comprising any one of SEQ ID NOs: 148-383 and 621-638.

18. The complex of embodiment 17, wherein the oligonucleotide comprisesa sequence comprising any one of SEQ ID NOs: 159, 162, 172, 174, 180,182, 188, 190, 195, 196, 201, 203, 212, 215, 218, 222, 248, and 264.

18.1. The complex of embodiment 18, wherein the oligonucleotidecomprises a sequence comprising any one of SEQ ID NOs: 180, 212, and222.

19. The complex of any one of embodiments 1-14, wherein theoligonucleotide comprises a region of complementarity to any one of SEQID NO: 384-619.

20. The complex of embodiment 19, wherein the oligonucleotide comprisesa region of complementarity to at least 15 consecutive nucleotides ofany one of SEQ ID NO: 384-619.

21. The complex of any one of embodiments 15 to 20, wherein theoligonucleotide comprises a region of complementarity to the DMPK allelecomprising the disease-associated-repeat expansion.

22. The complex of any one of embodiments 1 to 14, wherein the molecularpayload is a polypeptide.

23. The complex of embodiment 22, wherein the polypeptide is amuscleblind-like (MBNL) polypeptide.

24. The complex of any one of embodiments 15 to 21, wherein theoligonucleotide comprises an antisense strand that hybridizes, in acell, with a wild-type DMPK mRNA transcript encoded by the allele,wherein the DMPK mRNA transcript comprises repeating units of a CUGtrinucleotide sequence.

25. The complex of any one of embodiments 15 to 21, wherein theoligonucleotide comprises an antisense strand that hybridizes, in acell, with a mutant DMPK mRNA transcript encoded by the allele, whereinthe DMPK mRNA transcript comprises repeating units of a CUGtrinucleotide sequence.

26. The complex of any one of embodiments 1 to 25, wherein thedisease-associated-repeat is 38 to 200 repeating units in length. 27.The complex of embodiment 26, wherein the disease-associated-repeat isassociated with late onset myotonic dystrophy.

28. The complex of any one of embodiments 1 to 25, wherein thedisease-associated-repeat is 100 to 10,000 repeat units in length.

29. The complex of embodiment 28, wherein the disease-associated-repeatis associated with congenital myotonic dystrophy.

30. The complex of any one of embodiments 15 to 21 and 24 to 29, whereinthe oligonucleotide comprises at least one modified internucleotidelinkage.

31. The complex of embodiment 30, wherein the at least one modifiedinternucleotide linkage is a phosphorothioate linkage.

32. The complex of embodiment 31, wherein the oligonucleotide comprisesphosphorothioate linkages in the Rp stereochemical conformation and/orin the Sp stereochemical conformation.

33. The complex of embodiment 32, wherein the oligonucleotide comprisesphosphorothioate linkages that are all in the Rp stereochemicalconformation or that are all in the Sp stereochemical conformation.

34. The complex of any one of embodiments 15 to 21 and 24 to 33, whereinthe oligonucleotide comprises one or more modified nucleotides.

35. The complex of embodiment 34, wherein the one or more modifiednucleotides are 2′-modified nucleotides.

36. The complex of any one of embodiments 15 to 21 and 24 to 35, whereinthe oligonucleotide is a gapmer oligonucleotide that directs RNAseH-mediated cleavage of a DMPK mRNA transcript in a cell.

37. The complex of embodiment 36, wherein the gapmer oligonucleotidecomprises a central portion of 5 to 15 deoxyribonucleotides flanked bywings of 2 to 8 modified nucleotides.

38. The complex of embodiment 37, wherein the modified nucleotides ofthe wings are 2′-modified nucleotides.

39. The complex of any one of embodiments 15 to 21 and 24 to 35, whereinthe oligonucleotide is a mixmer oligonucleotide.

40. The complex of embodiment 39, wherein the mixmer oligonucleotideinhibits binding of muscleblind-like protein 1, muscleblind-like protein2, or muscleblind-like protein 3 to the DMPK mRNA transcript.

41. The complex of embodiment 39 or 40, wherein the mixmeroligonucleotide comprises two or more different 2′ modified nucleotides.

42. The complex of any one of embodiments 15 or 21 and 24 to 35, whereinthe oligonucleotide is an RNAi oligonucleotide that promotesRNAi-mediated cleavage of the DMPK mRNA transcript.

43. The complex of embodiment 42, wherein the RNAi oligonucleotide is adouble-stranded oligonucleotide of 19 to 25 nucleotides in length.

44. The complex of embodiment 42 or 43, wherein the RNAi oligonucleotidecomprises at least one 2′ modified nucleotide.

45. The complex of any one of embodiments 35, 38, 41, or 44, whereineach 2′ modified nucleotide is selected from the group consisting of:2′-O-methyl, 2′-fluoro (2′-F), 2′-O-methoxyethyl (2′-MOE), and 2′,4′-bridged nucleotides.

46. The complex of embodiment 34, wherein the one or more modifiednucleotides are bridged nucleotides.

47. The complex of any one of embodiment 35, 38, 41, or 44, wherein atleast one 2′ modified nucleotide is a 2′,4′-bridged nucleotide selectedfrom: 2′,4′-constrained 2′-O-ethyl (cEt) and locked nucleic acid (LNA)nucleotides.

48. The complex of any one of embodiments 15 to 21 and 24 to 35, whereinthe oligonucleotide comprises a guide sequence for a genome editingnuclease.

49. The complex of any one of embodiments 15 to 21 and 24 to 35, whereinthe oligonucleotide is phosphorodiamidite morpholino oligomer.

50. The complex of any one of embodiments 1 to 49, wherein themuscle-targeting agent is covalently linked to the molecular payload viaa cleavable linker.

51. The complex of embodiment 50, wherein the cleavable linker isselected from: a protease-sensitive linker, pH-sensitive linker, andglutathione-sensitive linker.

52. The complex of embodiment 51, wherein the cleavable linker is aprotease-sensitive linker.

53. The complex of embodiment 52, wherein the protease-sensitive linkercomprises a sequence cleavable by a lysosomal protease and/or anendosomal protease.

54. The complex of embodiment 52, wherein the protease-sensitive linkercomprises a valine-citrulline dipeptide sequence.

55. The complex of embodiment 51, wherein the linker is pH-sensitivelinker that is cleaved at a pH in a range of 4 to 6.

56. The complex of any one of embodiments 1 to 49, wherein themuscle-targeting agent is covalently linked to the molecular payload viaa non-cleavable linker.

57. The complex of embodiment 56, wherein the non-cleavable linker is analkane linker.

58. The complex of any of embodiments 2 to 57, wherein the antibodycomprises a non-natural amino acid to which the oligonucleotide iscovalently linked.

59. The complex of any of embodiments 2 to 57, wherein the antibody iscovalently linked to the oligonucleotide via conjugation to a lysineresidue or a cysteine residue of the antibody.

60. The complex of embodiment 59, wherein the antibody is conjugated tothe cysteine via a maleimide-containing linker, optionally wherein themaleimide-containing linker comprises a maleimidocaproyl ormaleimidomethyl cyclohexane-1-carboxylate group.

61. The complex of any one of embodiments 2 to 60, wherein the antibodyis a glycosylated antibody that comprises at least one sugar moiety towhich the oligonucleotide is covalently linked.

62. The complex of embodiment 61, wherein the sugar moiety is a branchedmannose.

63. The complex of embodiment 61 or 62, wherein the antibody is aglycosylated antibody that comprises one to four sugar moieties each ofwhich is covalently linked to a separate oligonucleotide.

64. The complex of embodiment 61, wherein the antibody is afully-glycosylated antibody.

65. The complex of embodiment 61, wherein the antibody is apartially-glycosylated antibody.

66. The complex of embodiment 65, wherein the partially-glycosylatedantibody is produced via chemical or enzymatic means.

67. The complex of embodiment 65, wherein the partially-glycosylatedantibody is produced in a cell, cell that is deficient for an enzyme inthe N- or O-glycosylation pathway.

68. A method of delivering a molecular payload to a cell expressingtransferrin receptor, the method comprising contacting the cell with thecomplex of any one of embodiments 1 to 67.

69. A method of inhibiting activity of DMPK in a cell, the methodcomprising contacting the cell with the complex of any one ofembodiments 1 to 67 in an amount effective for promoting internalizationof the molecular payload to the cell.

70. The method of embodiment 69, wherein the cell is in vitro.

71. The method of embodiment 69, wherein the cell is in a subject.

72. The method of embodiment 71, wherein the subject is a human.

73. The method of any one of embodiments 69 to 72, wherein the complexinhibits the expression of DMPK.

74. The method of any one of embodiments 69 to 73, wherein the cell iscontacted with a single dose of the complex.

75. The method of embodiment 74, wherein a single dose of the complexinhibits the expression of DMPK for at least two, four, eight, or twelveweeks.

76. The method of embodiment 75, wherein the complex inhibits theexpression of DMPK by at least 30%, 40%, 50%, or 60% relative to acontrol.

77. The method of embodiment 75 or 76, the complex inhibits theexpression of DMPK in muscle tissues by 40-60% for at least 12 weeksfollowing administration of the single dose, relative to a control

78. A method of treating a subject having an expansion of adisease-associated-repeat of a DMPK allele that is associated withmyotonic dystrophy, the method comprising administering to the subjectan effective amount of the complex of any one of embodiments 1 to 67.

79. The method of embodiment 78, wherein the disease-associated-repeatcomprises repeating units of a trinucleotide sequence.

80. The method of embodiment 78, wherein the trinucleotide sequence is aCTG trinucleotide sequence.

81. The method of any one of embodiments 78 to 80, wherein thedisease-associated-repeat is 38 to 200 repeating units in length.

82. The method of 81, wherein the disease-associated-repeat isassociated with late onset myotonic dystrophy.

83. The method of any one of embodiments 78 to 80, wherein thedisease-associated-repeat is 100 to 10,000 repeating units in length.

84. The method of 83, wherein the disease-associated-repeat isassociated with congenital myotonic dystrophy.

85. The method of any one of embodiments 78 to 84, whereinadministration of the complex results in inhibition of the expression ofDMPK in muscle tissues.

86. The method of any one of embodiments 78 to 85, wherein the complexis intravenously administered to the subject.

87. The method of any one of embodiments 78 to 86, wherein an effectiveamount of the complex comprises 1-15 mg/kg of RNA.

88. The method of any one of embodiments 78 to 87, wherein the complexis administered to the subject in a single dose. 89. The method ofembodiment 88, wherein administration of a single dose of the complexresults in inhibition of the expression of DMPK in muscle tissues for atleast two, four, eight, or twelve weeks.

90. The method of embodiment 89, wherein the administration of a singledose of the complex results in inhibition of the expression of DMPK inmuscle tissues by at least 30%, 40%, 50%, or 60% relative to a control.

91. The method of embodiment 89 or 90, wherein the administration of asingle dose of the complex results in inhibition of the expression ofDMPK in muscle tissues for at least 12 weeks following administration ofthe single dose.

92. The method of embodiment 91, wherein the administration of a singledose of the complex results in inhibition of the expression of DMPK inmuscle tissues by 40-60%, relative to a control, for at least 12 weeksfollowing administration of the single dose.

93. The method of embodiment 89 or 90, wherein the administration of asingle dose of the complex results in inhibition of the expression ofDMPK in muscle tissues for a duration of time in the range of 4-8, 5-10,8-12, 10-14, or 8-16 weeks following administration of the single dose.

94. The method of embodiment 93, wherein the administration of a singledose of the complex results in inhibition of the expression of DMPK inmuscle tissues by 40-60%, relative to a control, for a duration of timein the range of 4-8, 5-10, 8-12, 10-14, or 8-16 weeks followingadministration of the single dose.

95. The method of embodiment 89 or 90, wherein the administration of asingle dose of the complex results in inhibition of the expression ofDMPK in muscle tissues by 40-60%, relative to a control, at 12 weeksfollowing administration of the single dose.

96. The method of any one of embodiments 78 to 87, wherein the complexis administered to the subject in a single dose once every 4-8, 5-10,8-12, or 8-16 weeks.

97. The method of embodiment 96, wherein the complex is administered tothe subject in a single dose once every 12 weeks.

98. The method of any one of embodiments 88 to 97, wherein the singledose comprises the complex at a concentration of 1-15 mg/kg of RNA.

99. The method of embodiment 98, wherein the single dose comprises thecomplex at a concentration of 10 mg/kg of RNA.

100. A method of treating a subject having an expansion of adisease-associated-repeat of a DMPK allele that is associated withmyotonic dystrophy, the method comprising administering the complex ofany one of embodiments 1 to 67 to the subject,

wherein the administration results in inhibition of DMPK expression inmuscle tissues by 40-60%, relative to a control, for a duration of timein the range of 4-8, 5-10, 8-12, 10-14, or 8-16 weeks followingadministration of the complex.

101. A method of inhibiting DMPK expression in a subject, the methodcomprising administering the complex of any one of embodiments 1 to 67,

wherein the administration results in inhibition of DMPK expression inmuscle tissues by 40-60%, relative to a control, for a duration of timein the range of 4-8, 5-10, 8-12, 10-14, or 8-16 weeks followingadministration of the complex.

102. The method of embodiment 100 or 101, wherein the molecular payloadis an oligonucleotide.

103. The method of embodiment 102, wherein the concentration of thecomplex is 1-15 mg/kg of RNA.

104. A complex comprising an anti-transferrin receptor (TfR) antibodycovalently linked to a molecular payload configured for reducingexpression or activity of DMPK, wherein the anti-TfR antibody comprises:

(i) a heavy chain variable region (VH) comprising an amino acid sequenceat least 85% identical to SEQ ID NO: 76; and/or a light chain variableregion (VL) comprising an amino acid sequence at least 85% identical toSEQ ID NO: 75;

(ii) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 69; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 70;

(iii) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 71; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 70;

(iv) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 72; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 70;

(v) a heavy chain variable region (VH) comprising an amino acid sequenceat least 85% identical to SEQ ID NO: 73; and/or a light chain variableregion (VL) comprising an amino acid sequence at least 85% identical toSEQ ID NO: 74;

(vi) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 73; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 75;

(vii) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 76; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 74;

(viii) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 77; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 78;

(ix) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 79; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 80; or

(x) a heavy chain variable region (VH) comprising an amino acid sequenceat least 85% identical to SEQ ID NO: 77; and/or a light chain variableregion (VL) comprising an amino acid sequence at least 85% identical toSEQ ID NO: 80.

105. A complex comprising an anti-transferrin receptor (TfR) antibodycovalently linked to a molecular payload configured for reducingexpression or activity of DMPK, wherein the anti-TfR antibody hasundergone pyroglutamate formation resulting from a post-translationalmodification.

EQUIVALENTS AND TERMINOLOGY

The disclosure illustratively described herein suitably can be practicedin the absence of any element or elements, limitation or limitationsthat are not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof”, and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the disclosure. Thus, it should be understood that although thepresent disclosure has been specifically disclosed by preferredembodiments, optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this disclosure.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups or other grouping of alternatives, thoseskilled in the art will recognize that the disclosure is also therebydescribed in terms of any individual member or subgroup of members ofthe Markush group or other group.

It should be appreciated that, in some embodiments, sequences presentedin the sequence listing may be referred to in describing the structureof an oligonucleotide or other nucleic acid. In such embodiments, theactual oligonucleotide or other nucleic acid may have one or morealternative nucleotides (e.g., an RNA counterpart of a DNA nucleotide ora DNA counterpart of an RNA nucleotide) and/or (e.g., and) one or moremodified nucleotides and/or (e.g., and) one or more modifiedinternucleotide linkages and/or (e.g., and) one or more othermodification compared with the specified sequence while retainingessentially same or similar complementary properties as the specifiedsequence.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Embodiments of this invention are described herein. Variations of thoseembodiments may become apparent to those of ordinary skill in the artupon reading the foregoing description.

The inventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A composition comprising complexes, wherein each complex comprises an anti-transferrin receptor (TfR) antibody covalently linked to at least one oligonucleotides, wherein the antibody is a Fab and comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 101 and a light chain comprising the amino acid sequence of SEQ ID NO: 90, wherein each anti-TfR antibody of the complexes is on average covalently linked to 1 to 3 oligonucleotides, and wherein the oligonucleotide targets a Dystrophia Myotonica Protein Kinase (DMPK) RNA and comprises a region of complementarity to a sequence as set forth in any one of SEQ ID NO: 384-619, wherein the region of complementarity is 15-25 nucleotides in length.
 2. The composition of claim 1, wherein the heavy chain of the antibody comprises an N-terminal pyroglutamate.
 3. The composition of claim 1, wherein the oligonucleotide comprises a 5′-X—Y—Z-3′ formula, wherein X and Z are flanking regions comprising one or more 2′-modified nucleosides selected from the group consisting of: 2′-O-methyl, 2′-fluoro, 2′-O-methoxyethyl, 2′,4′-bridged nucleosides, and a combination thereof, and wherein Y is a gap region and each nucleoside in Y is a 2′-deoxyribonucleoside.
 4. The composition of claim 1, wherein the oligonucleotide is 15-30 nucleotides in length.
 5. The composition of claim 1, wherein the oligonucleotide comprises at least 15 consecutive nucleotides of a sequence set forth in any one of SEQ ID NOs: 148-383, wherein any one or more of the thymine bases (T's) in the oligonucleotide is optionally a uracil base (U).
 6. The composition of claim 1, wherein the oligonucleotide comprises one or more phosphorothioate internucleoside linkages.
 7. The composition of claim 1, wherein the antibody is covalently linked to each oligonucleotide via a linker.
 8. The composition of claim 7, wherein the linker comprises a cleavable linker.
 9. The composition of claim 8, wherein the linker comprises a valine-citrulline sequence.
 10. The composition of claim 9, wherein each complex comprises a structure of:

wherein n is 3 and m is 4, wherein L1 comprises a spacer that is a substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, —O—, —N(R^(A))—, —S—, —C(═O)—, —C(═O)O—, —C(═O)NR^(A)—, —NR^(A)C(═O)—, —NR^(A)C(═O)R^(A)—, —C(═O)R^(A)—, —NR^(A)C(═O)O—, —NR^(A)C(═O)N(R^(A))—, —OC(═O)—, —OC(═O)O—, —OC(═O)N(R^(A))—, —S(O)₂NR^(A)—, —NR^(A)S(O)₂—, or a combination thereof, wherein each R^(A) is independently hydrogen or substituted or unsubstituted alkyl.
 11. The composition of claim 10, wherein L1 comprises


12. The composition of claim 1, wherein the region of complementarity is to a sequence as set forth in SEQ ID NO:
 395. 13. The composition of claim 1, wherein the region of complementarity is to a sequence as set forth in SEQ ID NO:
 398. 14. The composition of claim 1, wherein the region of complementarity is to a sequence as set forth in SEQ ID NO:
 408. 15. The composition of claim 1, wherein the region of complementarity is to a sequence as set forth in SEQ ID NO:
 410. 16. The composition of claim 1, wherein the region of complementarity is to a sequence as set forth in SEQ ID NO:
 416. 17. The composition of claim 1, wherein the region of complementarity is to a sequence as set forth in SEQ ID NO:
 418. 18. The composition of claim 1, wherein the region of complementarity is to a sequence as set forth in SEQ ID NO:
 424. 19. The composition of claim 1, wherein the region of complementarity is to a sequence as set forth in SEQ ID NO:
 426. 20. The composition of claim 1, wherein the region of complementarity is to a sequence as set forth in SEQ ID NO:
 431. 21. The composition of claim 1, wherein the region of complementarity is to a sequence as set forth in SEQ ID NO:
 432. 22. The composition of claim 1, wherein the region of complementarity is to a sequence as set forth in SEQ ID NO:
 437. 23. The composition of claim 1, wherein the region of complementarity is to a sequence as set forth in SEQ ID NO:
 439. 24. The composition of claim 1, wherein the region of complementarity is to a sequence as set forth in SEQ ID NO:
 448. 25. The composition of claim 1, wherein the region of complementarity is to a sequence as set forth in SEQ ID NO:
 451. 26. The composition of claim 1, wherein the region of complementarity is to a sequence as set forth in SEQ ID NO:
 454. 27. The composition of claim 1, wherein the region of complementarity is to a sequence as set forth in SEQ ID NO:
 458. 28. The composition of claim 1, wherein the region of complementarity is to a sequence as set forth in SEQ ID NO:
 484. 29. The composition of claim 1, wherein the region of complementarity is to a sequence as set forth in SEQ ID NO:
 500. 30. The composition of claim 1, wherein the oligonucleotide comprises at least 15 consecutive nucleotides of a sequence set forth in any one of SEQ ID NOs: 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212, 215, 218, 222, 248, and 264, wherein any one or more of the thymine bases (T's) in the oligonucleotide is optionally a uracil base (U). 